AAV capsid modifications that enable improved CNS-wide gene delivery through interactions with the transferrin receptor

Abstract

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. Applicants engineered an AAV capsid, BI-hTFRl, that binds human Transferrin Receptor (TfRl), a protein expressed on the blood-brain barrier (BBB). BI-hTFRl was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFRl substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9.

Description

AAV CAPSID MODIFICATIONS THAT ENABLE IMPROVED CNS-WIDE GENEDELIVERY THROUGH INTERACTIONS WITH THE TRANSFERRIN RECEPTORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 621,839,filed January 17, 2024. The entire content of the above-identified application is herebyincorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Nos. NS111689 andMH120096 awarded by the National Institutes of Health (NIH). The government has certain rightsin the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] Reference is made to the electronic sequence listing ("BROD-5975WP_ST26.xml";Size is 2,318,176 bytes, created on January 16, 2025), which is herein incorporated by referencein its entirety.REFERENCE TO ELECTRONIC TABLES

[0004] Reference is also made to Tables 1-2, filed on January 17, 2024, in the United StatesPatent and Trademark Office and assigned Serial No. 63 / 621,839. The Tables are hereinincorporated by reference in their entirety.TECHNICAL FIELD

[0005] The subject matter disclosed herein relates generally to enhancing the transduction ofan engineered AAV capsid into cells of the central nervous system (CNS) through interaction withthe transferrin receptor expressed on the surface of the cells. In particular, in examples describedherein, at least one of the capsid's proteins is modified to include an n-mer motif. Further examplesrelate to a vector system having one or more vectors for the expression of the modified AAV capsidproteins in a cell, the packaging of a recombinant nucleic acid cargo into the engineered AAVcapsids, and a method of delivering the cargo to the CNS by either administering the AAV capsidsdirectly to a host organism in vivo or transducing cells with the AAV capsids ex vivo andtransplanting the cells back into the host organism.1BACKGROUND

[0006] The development of gene therapies for neurodevelopmental and neurological disordershas been constrained by the inability to deliver transgenes throughout the CNS. Several studieshave reported engineered AAV9 capsids, most notably the AAV-PHP.B family, capable of highlyeffective gene transfer throughout the CNS after intravenous administration in adult mice.However, none of the engineered AAV capsids that cross the blood-brain barrier (BBB) andtransduce the mouse brain with high efficiency exhibit enhanced CNS tropism in primates. Thereis an unmet need for AAVs with enhanced transduction efficiency.SUMMARY

[0007] In an aspects, the techniques described herein relate to an engineered adeno associatedvirus (AAV) capsid polypeptide, including a transferrin receptor (TfR1) binding modificationsequence defined by the formula X1-X2-X3-[7-mer]-X4-X5-X6-X7, wherein the 7-mer is one ofthe amino acid sequences YSRIGPN (SEQ ID NO: 1390), YSRNSDN (SEQ ID NO: 1391),LHRLGPN (SEQ ID NO: 1392), FRSTNGV (SEQ ID NO: 1393), FVSTNGV (SEQ ID NO:1394), FZ1STNGZ2 (SEQ ID NO: 1395), FRSTNGZ3 (SEQ ID NO: 1396), or VESTNGR (SEQID NO: 1397), and wherein the 7-mer is inserted between amino acids 588 and 589 of an AAV9capsid polypeptide (e.g., SEQ ID NO: 1), or in an analogous position of a capsid polypeptide ofanother AAV serotype, and wherein X1, X2, X3, Х4, Х5, X6, X7 indicate an amino acidsubstitution at one or more of these positions in the capsid polypeptide flanking the inserted 7-mer(i.e., residues 586, 587, 588, 589, 590, 591 and 592 of AAV9 or corresponding positions of anyother AAV serotypes).

[0008] In an embodiment, the 7-mer includes one of YSRIGPN (SEQ ID NO: 1390),YSRNSDN (SEQ ID NO: 1391), or LHRLGPN (SEQ ID NO: 1392), and wherein X1 is A, G, E,L, N, Q, S, W, or M; X2 includes A, F, I, L, M, N, Q, P, T, V, or Y; X3 includes Q; X4 includesany amino acid; X5 includes D, F, G, I, L, M, Q, P, S, T, or V; X6 includes A, C, F, G, H, I, P, S,T, V, W, or Y; X7 includes D, E, Q, or T; or any combination thereof where there is at least oneamino acid substitution relative to the wild type sequence. In an embodiment, the TfR1 bindingmodification sequence includes or consists ofthe amino acid sequence of one of SEQ ID Nos: 60-1210.2

[0009] In an embodiment, the n-mer includes the amino acid sequence YSRIGPN (SEQ IDNO: 1390), and wherein X1 is A, G, E, L, N, Q, S, W, or M; X2 is A, F, I, L, M, N, Q, P, T, V, orY; X3 is Q; X4 is A, E, F, H, I, L, M, N, P, Q, V, Y, D, or G; X5 is D, F, G, I, L, M, Q, P, S, T, orV; X6 is A, C, F, G, H, I, P, S, T, V, W, or Y; X7 is D, E, Q, or T; or any combination thereofwhere there is at least one amino acid substitution relative to the wild type sequence. In anembodiment, the TfR1 binding modification sequence includes or consists of the amino acidsequence of one of SEQ ID Nos: 60-1136.

[0010] In an embodiment, the n-mer is YSRNSDN (SEQ ID NO: 1391), and X1 is S or W; X2is V, I, or F; X3 is Q; X4 is any amino acid; X5 is Q or T; X6 is A; X7 is Q; or any combinationthereof. In an embodiment, the TfR1 binding modification includes or consists of the amino acidsequence of one of SEQ ID Nos: 1152-1157.

[0011] In an embodiment, the n-mer is LHRLGPN (SEQ ID NO: 1392), and X1 is S, A, L, orM; X2 is A or P; X3 is Q; X4 is A, E, F, H, I, L, M, N, P, Q, V, or Y; X5 is Q; X6 is A, P, S, or T;X7 is D, E, Q, or T; or any combination thereof. In an embodiment, the TfR1 binding modificationsequence includes or consists of the amino acid sequence of one of SEQ ID Nos: 1158-1210.

[0012] In an embodiment, the 7-mer is FRSTNGV (SEQ ID NO: 1393), FVSTNGV (SEQ IDNO: 1394), FZ1STNGZ2 (SEQ ID NO: 1395), FRSTNGZ3 (SEQ ID NO: 1396), or VESTNGR(SEQ ID NO: 1397) and wherein X1 is S; X2 is A, S, M, or D; X3 is F, H, I, L, M, N, Q, R, Y, D,or E; X4 is A, S, or M; X5 is Q or P; X6 is A, F, H, Q, or S; X7 is A, D, E, F, Q, S, or T; or anycombination thereof. In an embodiment, the TfR1 binding modification sequence includes orconsists of the amino acid sequence of one of SEQ ID Nos: 1211-1389.

[0013] In an embodiment, the n-mer is FRSTNGV (SEQ ID NO: 1393), and X1 is S; X2 is Aor S; X3 is D; X4 is A or S; X5 is Q or P; X6 is A, F, H, Q, or S; X7 is D, E, Q, or T; or anycombination thereof. In an embodiment, the TfR1 binding modification sequence includes orconsists of the amino acid sequence of one of SEQ ID Nos: 1211-1266.

[0014] In an embodiment, the n-mer is FVSTNGV (SEQ ID NO: 1394), and X1 is S; X2 is A,S, or M; X3 is Q, E, or D; X4 is A or M; X5 is Q or P; X6 is A; X7 is E; or any combinationthereof. In an embodiment, the TfR1 binding modification sequence includes or consists of theamino acid sequence of one of SEQ ID Nos: 1267-1298.

[0015] In an embodiment, the n-mer is FZ1STNGZ2 (SEQ ID NO: 1395) or FRSTNGZ3 (SEQID NO: 1396), and X1 is S; X2 is A or S; X3 is Q or D; X4 is A; X5 is Q; X6 is A; X7 is E; or any3combination thereof. In an embodiment, the TfR1 binding modification sequence includes orconsists of the amino acid sequence of one of SEQ ID Nos: 1299-1319.

[0016] In an embodiment, the 7-mer is VESTNGR (SEQ ID NO: 1397), and X1 is S; X2 is Sor D; X3 is F, H, I, L, M, N, Q, R, or Y; X4 is A; X5 is Q or P; X6 is A; X7 is A, D, E, F, Q, S, orT; or any combination thereof. In an embodiment, the TfR1 binding modification sequencecomprises or consists of the amino acid sequence of one of SEQ ID Nos: 1320-1388.

[0017] In an embodiment, Z1 is selected from the group consisting of A, D, H, N, Q, and S;Z2 is K or R; and Z3 is selected from the group consisting of L, M, and R.

[0018] In an embodiment, the TfR1 binding modification comprises or consists of the aminoacid sequence of EAQYSRIGPNNQAQ (SEQ ID NO: 170); SADFRSTNGVAQAE (SEQ ID NO:1215); or GAQYSRIGPNPQPE (SEQ ID NO: 243).

[0019] In an embodiment, the viral capsid protein is VP1, VP2, VP3, or a combination thereof.In an embodiment, the other AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10.

[0020] In some aspects, the techniques described herein relate to an engineered AAV particle,including any engineered AAV capsid polypeptide as described herein, and further includerecombinant AAV genome encoding a transgene.a

[0021] In an embodiment, the transgene encodes a therapeutic polypeptide, an antibody orfragment thereof, a siRNA, a CRISPR-Cas system, Transcription Activator-like Effector (TALE)-or Zinc Finger Protein (ZFP)-based transcriptional activator; repressor; or epigenomic silencer, aRNA encoding a partial gene fragment designed for transplacing into an endogenous RNA, one ormore transfer RNAs, or a component thereof, or an OMEGA system or any component thereof. Inan embodiment, the transgene is operably linked to a regulatory sequence that promotes expressionin the CNS

[0022] In one aspect, embodiments are directed to a pharmaceutical composition, includingany recombinant engineered AAV particle described herein and an acceptable carrier.

[0023] In another aspect, embodiments described herein provide a method of delivering apolypeptide or polynucleotide to the CNS of a subject comprising administering thepharmaceutical composition of any of those described herein to the subject. In an embodiment, thepharmaceutical composition is delivered at a dosage between 0.1 x 1012 vg / kg to 1 x 1014 vg / kg.In an embodiment, the dosage is between 0.1 x 1012 vg / kg to 100 x 1012 vg / kg. In an embodiment,4the dosage is between 1 x 1012 vg / kg to 10 x 1012 vg / kg. In an embodiment, the dosage is 5 x 1012vg / kg. In an embodiment the dosage is 2 x 1012 vg / kg. In an embodiment, the pharmaceuticalcomposition is administered systemically or directly to the CNS.

[0024] In one embodiment, a method of manufacturing a recombinant engineered AAVcomprises culturing mammalian cells comprising (1) a polynucleotide encoding the engineeredAAV capsid polypeptide of any of those as described herein, (2) a polynucleotide encoding arecombinant AAV genome including a transgene operably linked to a regulatory sequence andflanked by AAV ITR sequences, and optionally (3) a polynucleotide encoding adenoviral helpergenes, under conditions sufficient for the production of recombinant engineered AAV particles;and recovering the recombinant engineered AAV particles from said culture.

[0025] In another aspect, embodiments described herein relate to a host cell for the productionof recombinant engineered AAV particles, which comprises a polynucleotide encoding theengineered AAV capsid polypeptide of any of those described herein, a polynucleotide encodinga recombinant AAV genome including a transgene operably linked to a regulatory sequence andflanked by AAV ITR sequences, and optionally a polynucleotide encoding adenoviral helpergenes.

[0026] In another aspect, embodiments described herein relate to an AAV library including apopulation of variant engineered recombinant AAV particles wherein each member of thepopulation of variant engineered recombinant AAV particles includes a variant recombinant AAVcapsid polypeptide having a modification defined by the formula X1-X2-X3-[7-mer]-X4-X5-X6-X7, and wherein the 7-mer is inserted between amino acids 588 and 589 of an AAV9 capsidpolypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype, andwherein X1, X2, X3, X4, X5, X6, X7 indicate one or more modifications at amino acid positionsin the capsid polypeptide flanking the inserted 7-mer, wherein the modification has been selectedfor binding of TfR1 and / or increased tropism for the CNS relevant to a reference AAV particlewithout the modification. In an embodiment, the another AAV serotype is AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10.

[0027] In another aspect, embodiments described herein relate to a method of screening anAAV library for a recombinant AAV particle that binds Tfr1 and / or has increased tropism for theCNS. Said method comprises assaying any AAV library as described herein for increased binding5of Tfr1 and / or tropism for the CNS relative to an AAV vector with a reference capsid and selectingthose recombinant AAV vectors that have increased binding of Tfr1 and / or tropism for the CNS.

[0028] In another aspect, embodiments described herein relate to a method for training amachine learning algorithm, including (a) receiving, by at least one computing device, a pluralityof AAV capsid polypeptide sequences, includinga modification for binding to a TfR1; (b) training,by at least one computing device, with the plurality of AAV capsid polypeptide sequencesincluding a modification for binding to a TfR1, a transferrin receptor (TfR1) targeting machinelearning model; and (c) deploying, by at least one computing device, the TfR1 targeting machinelearning algorithm.

[0029] In an embodiment, the TfR1 targeting machine learning model is trained to identifyone or more sequences from the plurality of sequences with increased binding of Tfr1.

[0030] In an embodiment, the TfR1 targeting machine learning model is trained to identifyone or more sequences from the plurality of sequences that decrease the transduction of off-targettissues.

[0031] In an embodiment, the TfR1 targeting machine learning model is trained to identifyone or more sequences from the plurality of sequences that bind to an extracellular domain ofTFRC.

[0032] In an embodiment, the TfR1 targeting machine learning model is trained to identifyone or more sequences from the plurality of sequences that bind to an apical domain.

[0033] In an embodiment, the modification is defined by the formula X1-X2-X3-[7-mer]-X4-X5-X6-X7, wherein X1, X2, X3, X4, X5, X6, and X, indicate modifications at one or more aminoacid positions in the capsid polypeptide flanking the inserted 7-mer (that is, at each of X₁, X2, Х3,X4, Xs, X6, and X7, the amino acid may be the wild-type amino acid at that position or an aminoacid substitution).

[0034] In an embodiment, the training includes unsupervised, supervised, semi-supervised,reinforcement, transfer, incremental, and curriculum. The transferrin receptor (TfR1) targetingmachine learning model comprises of linear classifiers, logistic classifiers, random forest, artificialneural networks, matrix factorization, support vector machines, K-means clustering, or K-nearestneighbor.

[0035] In an embodiment, the transferrin receptor (TfR1)- targeting machine learning modelcomprises Boltzmann machines, Bayesian networks, autoregressive models, variational6autoencoders (VAEs), diffusion models, energy-based models, flow-based models, generativeadversarial networks (GANs), mixture models, hidden Markov models, or large language models(LLMs).

[0036] In an embodiment, the transferrin receptor (TfR1) targeting machine learning modelcomprises convolutional neural networks (CNNs), recurrent neural networks (RNNs), long shortterm memory models (LSTMs), gated recurrent units (GRUs), capsule networks, attentionmechanisms, or transformer networks.

[0037] In an embodiment, the transferrin receptor (TfR1) targeting machine learning model ispre-trained and further trained to predict transferrin receptor (TfR1) targeting by a pluralitysequences.of

[0038] These and other aspects, objects, features, and advantages of the example embodimentswill become apparent to those with ordinary skill in the art upon considering the following detaileddescription of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] An understanding of the features and advantages of the present invention will beobtained by reference to the following detailed description that sets forth illustrative embodiments,in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0040] FIG. 1A-1F - AAV9 can be programmed to bind human TfR1. (1A) An AAV9-based NNK capsid library of variants with random 7-mer insertions between VP1 residues 588-589 was screened for selective binding to human TfR1 in pull-down or cell binding assays.Individually produced human TfR1-binding variants carrying a single-stranded AAVCAG-NLSmScarlet-P2A-Luciferase-WPRE-SV40pA genome exhibited enhanced species-specific (1B)association with and (1C) transduction (Luciferase activity) of CHO cells stably expressing TFRC.Values reported are normalized to AAV9 in each cell line. Two-way ANOVA test to determinesignificant differences in transduction among five AAV variants on five CHO cell lines withAAV9 as the main comparison group for each cell line and using a Bonferroni multiple comparisoncorrection: **** and *** indicate p ≤0.0001 and ≤0.001, respectively; n = 3 replicates, error barsindicate ± SEM. The TfR1-binding variants exhibited enhanced (1D) association with and (1E)transduction (Luciferase activity) of human brain microvascular endothelial cells (hBMVEC) andhCMEC / D3 cells. Values are normalized to AAV9 in each cell line. One-way ANOVA test to7determine significant differences among five AAV variants with AAV9 as the comparison groupfor each cell line and using a Dunnett's multiple comparison correction: **** *** *** and * indicatep≤0.0001, ≤0.001, and ≤0.05, respectively; n = 4 replicates, error bars indicate ± SEM. (1F) Thebinding kinetics between each capsid and full-length human TfR1 were assessed by BLI. AAVXprobes were loaded with capsid, and human TfR1 was used as an analyte. Sensorgram curve fits(black dash-dot lines) were generated by applying global 2:1 exponential association and decaymodels.

[0041] FIG. 2A-2D – Human TfR1-targeted capsids transduce human brain endothelialcells via interactions with the apical domain of human TfR1. (2A) The plots show transduction(Luciferase, normalized RLU) of hCME / D3 cells incubated with 3 x 108 vg / mL of the indicatedAAV and the specified OKT9 or AF2474 antibody concentrations. Two-way ANOVA test todetermine significant differences in transduction with AAV9 or BI-hTFR1 and one of twoantibodies at different concentrations with the no antibody control for each condition as the maincomparison group and using a Bonferroni multiple comparison correction: **** indicates p≤0.0001; n = 3 replicates, error bars indicate ± SEM. (2B) The effect of Tf on BI-hTFR1 binding tofull-length human TfR1 was assessed by BLI. AAVX probes were loaded with AAV9 or BIhTFR1. Human TfR1 that had or had not been pre-incubated with 300 nM holo-Tf was used as ananalyte. 2:1 binding model curve fits (black dash-dot lines) are shown. (2C) Biotinylated holo-Tfwas immobilized on streptavidin-coated BLI probes (SA probe), introduced first into human TfR1,and then into a buffer with or without the OKT9 antibody, and finally into BI-hTFR1 virusparticles. Segments shaded in gray highlight the indicated association step. (2D) BI-hTFR1 orAAV2 was incubated with hCMEC / D3 cells at 50,000 vg / cell for one hour at 4°C, with or withoutOKT9 (1 µg / mL) or Tf (1 µg / mL), and immunostained for AAV and TfR1. Scale bars = 15 µm.AAV9 binding to hCMEC / D3 cells was rarely detected; therefore, AAV2 was used as a control(Fig. 9).

[0042] FIG. 3A-3C – BI-ҺTFR1 is efficiently endocytosed and actively transported acrosshuman brain endothelial cells. (3A) Schematic shows the pooled transwell BBB modelexperimental design. (3B) The vector genomes in the bottom chamber were quantified by qPCR.Two-way ANOVA with Bonferroni multiple comparison correction: **** and *** indicate p≤0.0001 and ≤ 0.001, respectively; n = 3 transwell replicates, error bars indicate ± SEM. (3C) BI8hTFR1 or AAV2 were incubated with hCMEC / D3 cells at 25,000 vg / cell for one hour at 37°C andstained for endosomal markers Rab5 and Rab7 and the AAVs. Scale bar = 15 µm.

[0043] FIG. 4A-4D – BI-hTFR1 efficiently delivers genes to the CNS of TFRC KI mice.(4A) In TFRC KI mice, mouse Tfrc exons 4-19 encoding the extracellular region of TfR1 havebeen replaced by those of human TFRC. (4B) BI-hTFR1 or AAV9 encoding CAG-NLS-mScarletP2A-Luciferase-WPRE-SV40pA were intravenously injected into adult female C57BL / 6J orTFRC KI mice at 5 x 1011 vg / mouse. AAV9 in C57BL / 6J and BI-hTFR1 in TFRC KI mice had n= 4 mice per group. AAV9 in TFRC KI and BI-hTFR1 in C57BL / 6J had n = 3 mice per group.The (4C) biodistribution reported as vector genomes per mouse genome and (4D) Luciferaseactivity within different organs are shown three weeks post-injection. Two-way ANOVA test todetermine significant differences within organs among the four groups of mice with BI-hTFR1 inTFRC KI mice as the main comparison group and using a Bonferroni multiple comparisoncorrection: **** ******, and * indicate p ≤ 0.0001, ≤ 0.001, ≤ 0.01, and ≤ 0.05, respectively;each data point represents an individual mouse, error bars indicate ± SEМ.

[0044] FIG. 5A-5F - BI-hTFR1 efficiently transduces neurons and astrocytes throughoutthe CNS. Representative (5A) whole brain and (5B) spinal cord images from each group of miceat three weeks post-injection are shown. Representative images show cells transduced by BIhTFR1 overlaid with (5C) NeuN+ or (5E) SOX9+ stained cells in the cortex, thalamus, and striatumof TFRC KI mice. The percentages of (5D) NeuN neurons or (5F) SOX9+ astrocytes thatexpressed mScarlet in the cortex, striatum, and thalamus are shown. Two-way ANOVA test todetermine significant differences in transduction within specific brain regions among the fourgroups of mice with BI-hTFR1 in TFRC KI mice as the main comparison group and using aBonferroni multiple comparison correction: **** indicates p≤ 0.0001; each data point representsan individual mouse, error bars indicate ± SEM.

[0045] FIG. 6A-6D – BI-hTFR1 efficiently delivered GBAI and increased GCase activityin the brains of TFRC KI mice. (6A) Schematic of the experiment shows the sSDNA AAVgenome expressing human glucocerebrosidase that was packaged into AAV9 or BI-hTFR1 andadministered to TFRC KI transgenic mice at either 1 x 1014 vg / kg or 5 x 1012 vg / kg. (6B) Thebiodistribution of AAV genomes found in brain and liver tissue relative to AAV9 (n = 4 mice percondition) is shown. One-way ANOVA with Sidak's multiple comparison post-hoc test usingAAV9 as the control group: **** and *** indicate p ≤ 0.0001 and ≤ 0.001; each data point9represents an individual mouse, and error bars indicate ± SEM. (6C) Sagittal brain sections (top)from the mice in each group show GBA-HA (magenta) in whole brain sagittal sections. Scale bar= 1 mm. Images of immunostaining (bottom) show neurons (NeuN, light gray) and GBA-НА(gray) in the substantia nigra pars compacta. Scale bar = 25 µm. (6D) GCase enzyme activity levelsin brain and liver tissue homogenate, CSF, and serum are shown. One-way ANOVA with Sidak'smultiple comparison post-hoc tests with AAV9 as the control group: **** indicates p ≤ 0.0001;each data point represents an individual mouse,and error bars indicate ± SEM.

[0046] FIG. 7A-7B - Validation assays confirm the expression of TfR1 from the indicatedspecies in stable CHO cell lines. (7A) Species-specific primers (table S1) were used to assessTFRC or Tfrc mRNA expression levels by RT-qPCR. Each species primer set is shown with theindicated color (legend, top). The graph shows qPCR cycle threshold (Ct) values showing selectiveearly cycle number amplification of the target species sequence. The bar indicates the mean Ctvalue (n = 3 replicates per cell line). (7B) Images show immunofluorescence for TfR1. Each cellline was fixed, permeabilized, and stained with the indicated anti-TfR1 antibodies to assess theexpression of exogenous TfR1.

[0047] FIG. 8A-8D - The binding between BI-hTFR1 and human TfR1 is inhibited bythe apical domain binding antibody OKT9 but not by holo-Tf. (8A) To assess binding betweeneach AAV capsid and full-length human TfR1, AAVx probes were loaded with ca,psid and TfR1was used as an analyte. Average kinetic constants and standard errors were computed fromtriplicate trials; * indicates a capsid for which values were computed from only two trials. (8B)The sensorgram shows the association of BI-hTFR1 to the Fc-hTfR1-loaded HFCII probe anddissociation in the presence of the indicated concentration of OKT9. The gray curve shows OKT9binding to Fc-hTfR1 in the absence of BI-hTFR1. (8C) The binding kinetics between full-lengthhuman TfR1 and human holo-Tf were assessed by BLI. Mouse Fc (MFC) probes were loaded withan anti-transferrin antibody, 12A6. Holo-Tf was loaded onto the probe and associated with serialdilutionthe s of TfR1 analyte. (8D) The effect of Tf on BI-hTFR1 binding to full-length humanTfR1 was assessed by BLI. AAVx probes were loaded with AAV9 or BI-hTFR1. Human TfR1that either had or had not been pre-incubated with 300 nM holo-Tf was used as an analyte. Averagekinetic constants and standard errors were computed from triplicate trials.

[0048] FIG. 9A-9C – AAV colocalizes with TfR1 and Tf in brain endothelial cells. (9A)The indicated AAVs were incubated with hCMEC / D3 cells at 25,000 vg / cell for one hour at 37°C.10Cells were then fixed, permeabilized, and stained for AAV (light gray) and nuclei (dark gray).AAV2 was chosen as a control for subsequent experiments because AAV9 immunostaining wasnearly undetectable. (9B) BI-hTFR1 or AAV2 was incubated with hCMEC / D3 cells at 30,000vg / cell for one hour at 37°C. Cells were fixed, permeabilized, and stained for AAV (light gray)and TfR1 (dark grey). (9C) BI-hTFR1 or AAV2 was incubated with hCMEC / D3 cells at 25,000vg / cell in prechilled media containing Tf-647 (1 µg / mL) for one hour at 4°C. Cells were then fixedand stained for AAV, Tf, and nuclei. All scale bars = 15 µm.

[0049] FIG. 10 - AAV colocalization with subcellular compartment markers. BI-hTFR1or AAV2 was incubated with hCMEC / D3 cells at 25,000 vg / cell for one hour at 37°C. Cells werethen fixed, permeabilized, and stained for AAV, endosomes (Rab5 and Rab7), cis-Golgi (RCAS1),trans-Golgi network (TGN46) or endoplasmic reticulum (KDEL (SEQ ID NO: 1787)), and nuclei.All scale bars = 15 µum.v

[0050] FIG. 11A-11B – TfR1 levels in TFRC KI mice match those in WT mice. (11A) RTqPCR using primers spanning exon 1 and exon 2 of the mouse Ifrc gene (table S1) was performedto assess mRNA levels in TFRC KI mice or C57BL / 6J controls treated with either AAV9 or BIhTFR1 (n = 3 or 4 animals per group; error bars indicate ± SEM). No significant difference inTfR1 mRNA between the C57BL / 6J and TFRC KI mice was detected in the cortex or spinal cord(two-way ANOVA). (11B) Western blots of protein from the brain tissues of C57BL / 6J and TFRCKI homozygous mice injected with AAV9 or BI-hTFR1 and purified full-length human or mouseTfR1 Fc-fusion proteins with N-terminal flag tag were stained using a polyclonal anti-human TfR1antibody, AF2474 (R&D systems), or a monoclonal antibody, H68.4 (Thermofisher, Catalog #13-6890) that recognizes mouse and human TfR1.

[0051] FIG. 12A-12C – BI-hTFR1 exhibited a CNS-specific enhanced tropism in TFRCKI mice and similar transduction to AAV9 in the liver and dorsal root ganglion. BI-hTFR1or AAV9:CAG-NLS-mScarlet-P2A-Luciferase-WPRE-SV40pA was intravenously injected intoadult female C57BL / 6J or TFRC KI mice at a dose of 5 x 1011 vg / mouse. The (12A) mScarlettranscript levels relative to AAV9 in C57BL / 6J mice and native fluorescence in the (12B) liverand (12C) dorsal root ganglion at three weeks post-injection are shown. For (12A), two-wayANOVA to determine significant differences within organs among the four groups of mice withBI-hTFR1 in 1FRC KI mice as the main comparison group and using a Bonferroni multiple11comparison correction: ****, ***, **, and * indicate p ≤ 0.0001, ≤ 0.001, ≤ 0.01, and ≤ 0.05,respectively; each data point represents an individual mouse, error bars indicate ± SEM.

[0052] FIG. 13A-13C – GBA-HA-expression in mice's liver and brain regions injectedwith AAV9 or BI-hTFR1:GBA1. (13A) Images show NeuN (light gray) and HA (dark gray)immunostaining in the deep cerebellar nuclei and brainstem in TFRC KI mice with the indicatedtreatment conditions. Scale bar = 25 µm. (13B) The cell counts of transduced NeuN+ cellsexpressing GBA-HA in the cortex, striatum, and thalamus of TFRC KI mice injected with 1 х 1014vg / kg BI-hTFR1:GBA1 are shown. Each data point represents a mouse (n = 4). Error bars indicate± SEM. (13C) Representative images of transduced liver hepatocytes stained for HA in theindicated conditions are shown. Scale bar = 50 µm.

[0053] FIG. 14A-14C - CNS transduction by example TfR1 binding AAVs. 5×1010vg / animal of ssAAV-CAG-NLS-mScarlet-2A-Luciferase-pA was administered into the retroorbital sinus of adult human TFRC knock-in mice and transduction was assessed 3 weeks postinjection. (14A) Images show native mScarlet fluorescence in sagittal brain sections from miceinjected with TfR1 binding capsid, BI-hTFR1 (top), a variant of BI294 (middle), or the BI295(bottom). (14B, C) The graphs show the fraction of neurons (14B) or astrocytes (14C) withdetectable mScarlet expression (n=3 per group).

[0054] FIG. 15A-15B - Peripheral organ transduction by example TfR1 binding AAVs.5x1010 vg / animal of ssAAV-CAG-NLS-mScarlet-2A-Luciferase-pA was administered into theretro-orbital sinus of adult TFRC knock-in mice and transduction was assessed 3 weeks postinjection. (15A) Images show native mScarlet fluorescence in liver sections from mice injectedwith TfR1 binding capsid, BI-hTFR1 (left), variant of BI294 (middle), or the BI295 (right). (15B)The graph shows the biodistribution assessment for AAV specific sequences (n=3 per group).

[0055] FIG. 16A-16D - Amino acid swaps between human and macaque TfR1 identifyspecific human residues that are required for BI294 and BI295 binding to TfR1. (16A) (SEQID NO: 1398-1400) An alignment of the human, macaque and marmoset amino acid sequencesfor the region containing the apical domain of TfR1 (AA 200 to 380). AAs that differ from thehuman TfR1 sequence are highlighted in blue. Four groupings of one to three surface exposedamino acids that differ between human and macaque are highlighted in outlined boxes in A and onthe predicted structural models in 16B. (16C-D) CHO cells were transiently transfected withplasmids carrying a cDNA encoding human TfR1, macaque TfR1, human TfR1 variants with12indicated amino acids replaced with the corresponding macaque amino acid sequence (left panels),or with macaque TFRC variants with the indicated amino acids replaced with the correspondinghuman amino acid (right panels). BI294:ssAAV-CAG-NLS-mScarlet-2A-Luciferase-pA (16C) orBI295:sSAAV-CAG-NLS-m Scarlet-2A-Luciferase-pA (16D) was added to CHO cells expressingthe indicated TFRC expression plasmid and transduction was assessed by measuring luciferaseactivity.

[0056] FIG. 17 – AAV9 capsid residues 539-605 aligned to other previously describedcapsids. (SEQ ID NO: 1401-1420) The 7-mer insertion site between AAV9 residue 588 and 589is shown. The black bars above the alignment highlight surrounding residues that were modifiedin this study. Corresponding residues in other example capsid sequences are outlined and residuesthat differ from AAV9 are shown in gray. Sequences were aligned using MUSCLE (SnapGene).

[0057] FIG. 18 – The SEQ ID 34 and surrounding AAV9 sequence (SEQ ID 45) impartsTfR1 binding into multiple AAV capsids. Control CHO cells or stable CHO cells expressinghuman TFRC (hTFRC) were transduced with SEQ ID#21 (AAV9 w / SEQ ID#34), or modifiedAAV7 capsids where the SEQ ID#30 was inserted between AA589 and 590 (SEQ ID 28; AAV7w / TfR1 targeting 7-mer) or where AA587-AA593 were replaced with SEQ ID#40, whichcomprises SEQ ID 30 and flanking amino acids from AAV9 (SEQ ID 29; AAV7 w / TfR1 targeting7-mer and surrounding sequence). Bar plot shows the mean transduction (relative light units) percondition. Individual data points represent transduction replicates (n=4).

[0058] FIG. 19 -Transferrin Receptor binding modification (SEQ ID NO: 1421-1433) -example insertions between residues 558 and 559 AAV9 VP1.

[0059] FIG. 20- Example serotype sequence alignment (SEQ ID NO: 1, 3-5, 7-11, 15-18).

[0060] FIG. 21 – Transferrin Receptor binding moiety insertion site in AAV9 VP1 capsid(SEQ ID NO: 1, 1434-1436).

[0061] FIG. 22A-22B – Example TfR1-binding capsids exhibit increased biodistributionto the brain and spinal cord with a decreased biodistribution to the liver relative to AAV9and BI-hTFR1 following IV administration into TFRC KI mice. ssAAV-CAG-NLS-mScarletP2A-ffLuciferase-WPRE-pA was packaged into the indicated capsid and administered to 10 to 16-week-old humanized TFRC KI mice. AAV genome (WPRE) biodistribution was assessed 21 dayslater. (22A). The graph shows AAV genome biodistribution normalized relative to the housekeeping gene GAPDH. (22B). The graph reports the data from 22A after normalization to AAV9.13The viral genomes detected from BI-hTFR1, BI-hTFR1v2, BI-hTFR1v3 and BI-hTFR2v2 are 64-fold, 107-fold, 105-fold, and 56-fold greater than AAV9, respectively, in the spinal cord. n=6animals per capsid (3 female and 3 males). Error bars indicate SEM.

[0062] FIG. 23A-23B - Example TfR1-binding capsids more efficiently transduce thebrain and spinal cord relative to AAV9 IV administration in TFRC KI mice. Transduction inthe indicated organ was assessed by measuring Luciferase activity (relative luminescence units(RLU) per microgram of protein lysate). (23A). The graph shows luciferase RLU / ug protein inlysates from the indicated organs by the indicated capsids (legend). (23B). The graph reports thedata from A expressed as the fold change in RLU per microgram of protein lysate relative toAAV9. n=6 animals per capsid (3 female and 3 males) of 10-16 weeks of age at time of injection.Error bars indicate + SEM.

[0063] FIG. 24 - The TFR1 binding capsids mediate efficient transduction across theCNS. ssAAV-CAG-NLS-mScarlet-P2A-ffLuciferase-WPRE-pA was packaged into the indicatedcapsid and administered to 10 to 16-week-old humanized TFRC KI mice. mScarlet expression wasassessed 21 days later. Images show native mScarlet (white) fluorescence from representativesagittal sections of B-hTFR1 mouse brains.

[0064] FIG. 25 – Example Capsid efficiently transduces neurons in the cortex of BhTFR1 mice. Adult (10- to 16-week-old) humanized TFRC KI mice (B-hTFR1) were dosed with2e12 vg / kg of ssCAG-NLS-mScarlet-P2A-fLuciferase-WPRE-рA рackaged into AAV9, AAVPHP.eB, BI-hTFR1, or BI-hTFR1v2. Representative images of native mScarlet fluorescence (top)and the immunofluorescence for the neuronal marker NeuN (bottom) in the cerebral cortex 21 dayspost administration are shown.

[0065] FIG. 26 - Example capsid demonstrates increased transduction of neurons thanBI-hTFR1 after IV delivery to humanized TFRC KI mice. Adult (10- to 16-week-old)humanized TFRC KI mice (B-hTFR1) were dosed with 2e12 vg / kg of ssCAG-NLS-mScarlet-P2АfLuciferase-WPRE-рA packaged into AAV-PHP.eB, BI-hTFR1, or BI-hTFR1v2 and thetransduction of NeuN positive neurons in the cortex or striatum was assessed 21 days later bymeasuring the fraction of the NeuN+ cells that expressed mScaret (native fluorescence). n=6animals per capsid (3 female and 3 males). Data points represent values from individual animals.Error bars indicate + SEM.14

[0066] The figures herein are for illustrative purposes only and are not necessarily drawn toscale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSDefinitions

[0067] Unless defined otherwise, technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which this disclosurepertains. Definitions of common terms and techniques in molecular biology may be found inMolecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis);Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); CurrentProtocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods inEnzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D.Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.):Antibodies A Laboratory Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture(1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nded., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions,Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H.Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0068] Titles or subtitles may be used in the specification for the sole convenience of the readerbut are not intended to influence the scope of the present disclosure or to limit any aspect of thedisclosure to any subsection, subtitle, or paragraph.

[0069] As used herein, the singular forms "a", "an", and "the" include both singular and pluralreferents unless the context clearly dictates otherwise. The term "optional" or "optionally" meansthat the subsequent described event, circumstance or substituent may or may not occur, and thatthe description includes instances where the event or circumstance occurs and instances where itdoes not.15

[0070] The recitation of numerical ranges by endpoints includes all numbers and fractionssubsumed within the respective ranges and the recited endpoints. For example, "1-5 ng" or a rangeof "1 ng to 5 ng" is intended to encompass 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 1-2 ng, 1-3 ng, 1-4 ng, 1-5ng, 2-3 ng, 2-4 ng, 2-5 ng, 3-4 ng, 3-5 ng, and 4-5 ng.

[0071] The terms "about" or "approximately" as used herein when referring to a measurablevalue such as a parameter, an amount, a temporal duration, and the like, are meant to encompassvariations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations areappropriate to perform in the disclosed invention. It is to be understood that the value to which themodifier "about" or "approximately" refers is itself also specifically and preferably, disclosed.

[0072] As used herein, a "biological sample" may contain whole cells and / or live cells and / orcell debris. The biological sample may contain (or be derived from) a "bodily fluid". The presentinvention 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, peritonealfluid, 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 samplesinclude cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtainedfrom a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0073] The terms "subject," "individual," and "patient" are used interchangeably herein torefer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but arenot 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 alsoencompassed.

[0074] As used herein, the phrase "at least one," in reference to a list of one or more elements,should be understood to mean at least one element selected from any one or more of the elementsin the list of elements, but not necessarily including at least one of each and every elementspecifically listed within the list of elements, and not excluding any combinations of elements inthe list of elements. This definition also allows that elements may optionally be present other thanthe elements specifically identified within the list of elements to which the phrase "at least one"16refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or,equivalently "at least one of A and / or B") can refer, in one aspect, to at least one, optionallyincluding more ore 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 (andoptionally including elements other than A); in yet another aspect, to at least one, optionallyincluding more than one, A, and at least one, optionally including more than one, B (and optionallyincluding other elements); etc.

[0075] As used herein, the terms "treat," "treatment," "treating" refer to therapeutic treatments,wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progressionor severity of a disorder. The term "treating" includes reducing or alleviating at least one adverseeffect or symptom of a condition, disease, or disorder. Treatment is generally "effective" if one ormore symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if theprogression of a disorder is reduced or halted. That is, "treatment" includes not just theimprovement of symptoms or markers, but also a cessation of, or at least slowing of, progress orworsening of symptoms compared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation of the disease state, remission(whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term"treatment" of a disease also includes providing relief from the symptoms or side-effects of thedisease (including palliative treatment).

[0076] The term "expression vector", as used herein, refers to a nucleic acid constructcomprising nucleic acid elements sufficient for the expression of a transgene product in a cell orin an in vitro assay. For example, a transgene expression vector, disclosed herein, comprises apromoter operatively linked to a transgene transcription unit comprising a transcription initiationsite, 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 polyadenylationsequence.

[0077] The term "operably linked" refers to a functional relationship between two or morepolynucleotide (e.g., DNA) segments. Typically, the term refers to the functional relationship of a17transcriptional regulatory sequence and a transgene to be transcribed. For example, a promoter orenhancer sequence is operably linked to a transgene if it, e g., stimulates or modulates the transgenetranscription in an appropriate host cell or other expression system. Generally, promotertranscriptional regulatory' sequences that are operably linked to a sequence are contiguous to thatsequence or are separated by short spacer sequences, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not be physically contiguous orlocated in close proximity to the coding sequences whose transcription they enhance.

[0078] As used herein, "recombinant" refers to nucleic acids or polypeptides that aregenetically engineered.

[0079] A "transgene" is a polynucleotide sequence that may encode an RNA (mRNA) that istranslated into protein. For example, a transgene may comprise a cDNA sequence. In otherembodiments, a transgene may encode on "non-coding" RNA that is not translated into protein (eg. guide RNAs, ribozymes, aptamers, antisense RNAs, piwi-interacting RNAs (piRNAs), shortinterfering RNAs (siRNAs), microRNAs (miRNAS), shRNAs or recombinant U RNAs). In someaspects, the transgene nucleotide sequence may comprise one or more introns. In other aspects, thetransgene can be polycistronic (e.g., two coding regions separated by internal ribosome entry site(IRES)). In some aspects, a transgene may encode more than one protein. In one aspect, atransgene comprises a "protein coding sequence" or a sequence that encodes a particular proteinor polypeptide, i.e., a nucleic acid sequence that is capable of being transcribed into mRNA andtranslated into a polypeptide in vitro or in vivo when placed under the control of appropriateregulatory sequences. The boundaries of the coding sequence may be determined by a start codonat the 5' terminus (N-terminus) and a translation stop nonsense codon at the 3' terminus (Cterminus). A coding sequence can include, but is not limited to, cDNA from prokaryotic oreukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and syntheticnucleic acids. A transcription termination sequence will usually be located 3' to the codingsequence. A "serotype" is traditionally defined on the basis of a lack of cross-reactivity betweenantibodies to one virus as compared to another virus. Such cross-reactivity differences are usuallydue to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2,and / or VP3 sequence differences of AAV serotypes). Under the traditional definition, a serotypemeans that the virus of interest has been tested against serum specific for all existing andcharacterized serotypes for neutralizing activity and no antibodies have been found that neutralize18the virus of interest. As more naturally occurring virus isolates are discovered and capsid mutantsgenerated, there may or may not be serological differences with any of the currently existingserotypes. Thus, in cases where the new AAV has no serological difference, this new AAV wouldbe a subgroup or variant of the corresponding serotype. In many cases, serology testing forneutralizing activity has yet to be performed on mutant viruses with capsid sequence modificationsto determine if they are of another serotype according to the traditional definition of serotype.Accordingly, for the sake of convenience and to avoid repetition, the term "serotype" broadlyrefers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are notserologically distinct that may be within a subgroup or a variant of a given serotype.

[0080] "Transduction" refers to the transfer of a transgene into a recipient host cell by a viralvector. Transduction of a target cell by an rAAV virion of the invention leads to transfer of thetransgene contained in that rAAV virion into the transduced cell. "Host cell" or "target cell" refersto the cell into which the DNA delivery takes place, such as the cells of the CNS or HEK293Tcells in case of the in vitro transduction assay. AAV vectors are able to transduce both dividingand non-dividing cells. In a cell comprising a gene product of interest, such as for example GFP,the gene product of interest has been introduced / transferred / transduced by rAAV "transduction"of the cell. A cell into which the transgene has been introduced is referred to as a "transduced"cell

[0081] Various embodiments are described hereinafter. It should be noted that the specificembodiments are not intended as an exhaustive description or as a limitation to the broader aspectsdiscussed herein. One aspect described in conjunction with a particular embodiment is notnecessarily limited to that embodiment and can be practiced with any other embodiment(s).Reference throughout this specification to “one embodiment", "an embodiment", "an exampleembodiment," means that a particular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of the present invention. Thus,appearances of the phrases "in one embodiment," "in an embodiment," or "an exampleembodiment" in various places throughout this specification are not necessarily all referring to thesame embodiment, but may. Furthermore, the particular features, structures or characteristics maybe combined in any suitable manner, as would be apparent to a person skilled in the art from thisdisclosure, in one or more embodiments. Furthermore, while some embodiments described hereininclude some but not other features included in other embodiments, combinations of features of19different embodiments are meant to be within the scope of the invention. For example, in theappended claims, any of the claimed embodiments can be used in any combination.OVERVIEW

[0082] Embodiments disclosed herein provide transferrin receptor binding modificationswhich promote transduction into the central nervous system (CNS) through its interaction with thecell surface transferrin receptor. These transferrin receptor binding modifications may beincorporated into particles, such as viral capsid delivery particles, including AAV9 particles andAAV particles of other serotypes, to confer tropism on the delivery particles and promotetransduction of CNS. Exemplary CNS tissues include brain and spinal cord tissue. Exemplary CNScell types include neurons, ependymal cells, and glial cells, e.g., microglia, astrocytes,oligodendrocytes, and NG2-glia progenitors, pericytes, as well as endothelial cells. Furtherembodiments disclosed herein provide for a vector system comprising one or more vectorsencoding AAV capsids according to embodiments described herein. Accordingly, embodimentsdisclosed herein provide compositions capable of delivering cargos with enhanced selectivity andefficiency to the CNS vasculature. Embodiments disclosed herein also provide vector systems forthe generation and loading of such delivery particles with cargo. Likewise, embodiments disclosedherein provide methods for use of such compositions to target CNS endothelial cells, in vitro andin vivo, with implications for both therapeutic and research purposes.

[0083] Additional features and advantages of the aforementioned embodiments are furtherdescribed below.Transferrin Receptor Binding Modifications and Compositions Thereof

[0084] In an embodiment, compositions are provided herein of AAV capsids comprisingcapsid proteins having a transferrin receptor binding modification sequence conferring on thecapsid an enhanced tropism for endothelial cells of the CNS. A transferrin receptor bindingmodification with an enhanced tropism for endothelial cells of the CNS promotes, increases, orotherwise improves binding to, and in some cases, transduction of the CNS as compared to anatural or wild-type target moiety. This transferrin receptor binding modification may be coupleddirectly to a cargo to be delivered, such as an oligonucleotide or polypeptide. Alternatively, thetargeting molecule may be incorporated into a delivery particle, such as an AAV particle (forexample, being incorporated into an AAV capsid protein) to confer tropism for endothelial cellsof the CNS on the delivery particle. A non-limiting example of a delivery particle is a viral capsid20particle. In such embodiments, the transferrin receptor binding modification may be incorporatedinto a viral capsid polypeptide such that the transferrin receptor binding modification isincorporated into the assembled viral capsid. However, other particle delivery systems where thetransferrin receptor binding modification may be incorporated or attached, for example onexosomes, liposomes, lipid nanoparticles, virus-like particles, ribonucleoproteins, nanobodies,antibodies, or antibody fragments are also envisioned and encompassed as alternativeembodiments herein.

[0085] In an embodiment, provided herein is a composition comprising a transferrin receptorbinding modification effective to increase transduction of CNS tissues via binding to a transferrinreceptor, optionally further comprising a cargo coupled to or otherwise associated with thetransferrin receptor binding modification. A transferrin receptor binding modification withincreased transduction promotes, enhances, or otherwise improves binding to and transduction ofthe CNS as compared to a natural or wild-type capsid without the transferrin receptor bindingmodifications described herein. In an example embodiment, the transferrin receptor bindingmodification binds to the extracellular domain of transferrin receptor (TFR). In an exampleembodiment, the transferrin receptor binding modification binds to one or more of the apical,helical, and / or protease-like domains of the extracellular domain. In an example embodiment, thetransferrin receptor binding modification binds to the apical domain. In an example embodiment,the n-mer is an amino acid sequence of length n. The length of the n-mer may be any necessarylength to transduce the CNS. In an embodiment, the n-mer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12amino acids in length. In an example embodiment, the n-mer motif has a length of at least 7 aminoacids. In an example embodiment, a composition comprising a targeting moiety effective toincrease transduction of CNS tissues comprises a n-mer motif, the n-mer motif comprising orconsisting of an amino acid sequence of Zı-X1-Z2-X2-X3-X4-Xs wherein Z1 is Y, F, or L, Z2 is S,R, or K, X1 - X5 are independently selected amino acids. In an example embodiment, X₁ optionallycomprises of A, S, or H; X2 optionally comprises of S, T, L, or I; X3 optionally comprises N or G;X4 optionally comprises G; Xs optionally comprises of N, D, I, V, or R. In an exampleembodiment, the targeting moiety comprises a n-mer motif, the n-mer motif comprising orconsisting of an amino acid sequence of is X1-H-X2-L-X3-X4-X5 wherein X1 X5 areindependently selected amino acids.21

[0086] In an embodiment, the n-mer can be used to increase transduction in target cells i.e.,CNS cells and tissues. The increase in transduction efficiency (which may correspond to thetropism efficiency) of the n-mer to a cell may be compared to a composition that does not containthe transferrin receptor binding modification for example inclusion of one or more transferrinreceptor binding modifications in a composition can result in an increase in transduction and ortransduction efficiency by 10%, 20%, 30%, 40%, 50%, 60% 70% 80% 90% a 100% or more. Inan exemplary embodiment the increase in transduction and or transduction efficiency is one and ahalf fold, two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, tenfold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold or more relative to a compositionlacking the n-mer. In one embodiment the transduction and / or transduction efficiency is increasedor enhanced in endothelial cells, in one embodiment increase in endothelial cells of the vasculature,for example, the central nervous system vasculature. In an embodiment, the transduction and / ortransduction efficiency is increased or enhanced in cells of the central nervous system. In anembodiment, the transduction and / or transduction efficiency is increased or enhanced in neuronsand glial cells In an embodiment, the composition comprising a n-mer is selective to a target cellas compared to other cell types and / or other virus particles. As used herein, 'selective' and 'cellselective' refers to preferential targeting for cells as compared to other cell types. Preferably, thetransferrin receptor binding modification is selective for a desired target (e.g., cell, organ, systeme.g., CNS tissues) or set of targets by at least 2:1, 3:1, 4:1, 5:1, 6:1 7:1, 8:1, 9:1. 10:1 or more; or10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% 80%, 85%, 90% or more, relative to other targets orcells (e.g., CNS). In an example embodiment, the composition comprising a Transferrin receptorbinding modification described herein can have an increased uptake, delivery rate, transductionrate, efficiency, amount, or a combination thereof in a target cell (e.g., endothelial cells across theCNS e.g., brain endothelium, arterio-venous axis in brain, retina, and spinal cord vasculature) ascompared to other cells types (e.g. hepatocytes) and / or other virus particles (e.g., AAVs notcontaining the transferrin receptor binding modification) and other compositions that do notcontain the cell-selective n-mer motif of the present invention.

[0087] In an example embodiment, the n-mer motif is selected from the group consisting ofYSRIGPN (SEQ ID NO: 1390), YSRLNMN (SEQ ID NO: 1437), YSRLNKD (SEQ ID NO:1438), and YHRLSNN (SEQ ID NO: 1439). In an example embodiment, the n-mer is VHRLQDK(SEQ ID NO: 1440) or LHALSHN (SEQ ID NO: 1441). In an example embodiment, the n-mer is22or comprises PSATNGV (SEQ ID NO: 1442), QVSTNGI (SEQ ID NO: 1443), SYSSNGV (SEQID NO: 1444), HQSSNGV (SEQ ID NO: 1445), VGSINGI (SEQ ID NO: 1446), AMSTNGR(SEQ ID NO: 1447), SASTNGV (SEQ ID NO: 1448), YMSTNGV (SEQ ID NO: 1449),YYSSNGV (SEQ ID NO: 1450), VHSTNGI (SEQ ID NO: 1451), PLSTNGV (SEQ ID NO: 1452),VYSTNGI (SEQ ID NO: 1453), IISTNGV (SEQ ID NO: 1454), RSVSSNGV (SEQ ID NO: 1455),YKSSNGV (SEQ ID NO: 1456), FRSTNGV (SEQ ID NO: 1393), and / or FVSTNGV (SEQ IDNO: 1394). In an example embodiment, the n-mer is selected from any of the amino acid sequencesin Tables 1-2 or any combination thereof. In an example embodiment, the transferrin receptorbinding modification is part of (e.g., inserted between consecutive amino acids of) a viral capsidprotein, including an AAV capsid protein. In an embodiment, the TfR1 binding modificationcomprises or consists of the amino acid sequence of EAQYSRIGPNNQAQ (SEQ ID NO: 170);SADFRSTNGVAQAE (SEQ ID NO: 1215); or GAQYSRIGPNPQPE (SEQ ID NO: 243).Transferrin Receptor (TFR) Binding

[0088] The role of the transferrin-transferrin receptor interaction is to transport iron into thevicinity of the cell surface, thereby increasing iron uptake. Transferrin is a blood plasma proteinfor iron ion delivery. Transferrin is a glycoprotein, which binds iron very tightly but reversibly.Although iron bound to transferrin is less than 0.1% (4 mg) of the total body iron, dynamically itrepresents the most important iron pool, with the highest rate of turnover (25 mg / 24 h). Transferrinhas a molecular weight of around 80 kDa and contains 2 specific high affinity Fe binding sites.The affinity of transferrin for Fe is extremely high (1023 M-1 at pH 7.4) but decreases progressivelywith decreasing pH below neutrality.

[0089] Transferrin- bound iron is delivered to a cell through a transferrin receptor-mediatedendocytosis. In vertebrates, there are two transferrin receptors. Transferrin receptor protein 1(TfR1), also known as Cluster of Differentiation 71, (CD71), P90, CD71, TRFR, Trfr, TFR, orIMD46, is a protein that in humans is encoded by the TFRC gene (Human TfR1 mRNA: AccessionNo. X01060.1). Human TFRC (HGNC:11763; Gene ID: ENSG00000072274), located onchromosome 3, is transcribed into 38 transcripts (splice variants), 284 orthologues, 5 paraloguesand is associated with 47 phenotypes.

[0090] Human TfR1 mediates cellular uptake of both transferrin and H-ferritin, an iron storageprotein. TfR1 is a transmembrane homodimer that consists of two identical monomers with amolecular mass of approximately 90 kDa (760 aa); each monomer is joined by two disulfide bonds23at Cys89 and Cys98. TfR1 further comprises a short, NH2-terminal cytoplasmic region (residues1 to 67), a single transmembrane pass (residues 68 to 88), and a large extracellular portion(ectodomain, residues 89 to 760), which is soluble and bears a trypsin-sensitive site and containsa binding site for transferrin. Synthesized in the endoplasmic reticulum, TfR1 is posttranslationally modified with both phosphate and fatty acyl groups. TfR1's extracellular domaincontains three N-linked glycosylation sites at Asn251, Asn317, and Asn727 and one O-linkedglycosylation site at Thr104. TfR1 is typically found as a dimer linked by disulfide bonds on thecell surface, see FIG. 4.

[0091] Human transferrin receptor 2 (TfR2), located on chromosome 7, is a protein that inhumans is encoded by the TFR2 gene (Gene Identifier: ENSG00000106327). TFR2 is lesscommonly expressed than TFRC and is predominantly expressed in hepatocytes.

[0092] The TFR1 contains an extracellular C-terminal domain (around 671 amino acids) andcomprises of the TF binding site. The extracellular C-terminal domain comprises of threesubdomains: apical, helical, and protease-like domain, see FIG. 4. Furthermore, the extracellularC-terminal domain comprises of three N-linked glycosylation sites at asparagine residues 251,317, and 727 and one O-linked glycosylation site at threonine 104, which contribute to adequatefunction of the receptor. TFR1 additionally comprises of a transmembrane domain (around 29amino acids), and an intracellular N-terminal domain (around 61 amino acids). Alternative to thedelivery of iron through TF with TFR1, uptake of iron can also occur through H-ferritin bindingto the apical domain. See also Candelaria, P. V., et al. Antibodies Targeting the TransferrinReceptor 1 (TfR1) as Direct Anti-Cancer Agents. Frontiers in Immunology, 2021, 12, herebyincorporated by reference.

[0093] In an example embodiment, the transferrin receptor binding modification binds to theextracellular domain of TFR1. In an example embodiment, the transferrin receptor bindingmodification binds to one or more of the helical (residues 606-760), the protease-like (residues121-183, 384-605) and the apical (residues 184-383) domain (Lawrence et al., Crystal structure ofthe ectodomain of human transferrin receptor. Science. 1999;286(5440):779-82). In anembodiment, the transferrin receptor binding modification binds to the apical domain. As usedherein, the term "a transferrin receptor" refers to TfR1 or TfR2 or any one of the alternativelyspliced variants, orthologues, paralogues. In an embodiment, the term "a transferrin receptor"refers to TfR1 or any one of the alternatively spliced variants, orthologues, paralogues.24Table A. Human Transferrin Receptor 1 cDNA sequence.HUMAN TRANSFERRIN RECEPTOR 1 cDNA SEQUENCE Location AMINOACID SEQ ID NO (AMINO ACID SEQ ID NO: 1459) Location DOMAINS (NUCLEOTIDE SEO ID NO: 1460)1MMDΟARSAFSNLFGGEPLSY 201 ATGATGGATCAAGCTAGATCAGCATTCTCTAACTTGTTTTGGGTTGCGAAGZAAACCAATTTGTCAATAT 6021TRFSLARΟVDGDNSHVEMKL 4061 ACCCGGTTCAGCCCTTGGCTCGGCAAGTAGATGGCGATAACAGTГСАATTGTGGAGATGAAACTT 12041AVDEENADNNTKANVTKPK 90NH2-TERMINALCYTOPLASMIC REGION(aa 1-67) 12 GCTGTAGATGAAGAAGAAAATGCTGAСААТААCACAAAGGCCAATGTCACAAAACCAAAA 18061 RC SGSTC (SEQ ID NO: 1461)181 AGGTGTAGTGGAAGTATCTGC (SEO ID NO: 1462)YGTIY 81 FMIG241 TTTATGATTGGCTGCTTGTAA GG101 LAGT301 CTGGCAGGAACCGAGTG121 RL Y361 CGCTTATATTGGGATGACCTGAAGAGAAAGTTGP Ε보F 141TSTI421 ACCAGCACCA161KDENA481 AAAGATGAAAATCTϋH1817WR(SEQ541 GTCTGGCGT (SEQDΟ201IVDKN601 ATCATAGTTGATAAGAACcב1AFGGATCAACATTAΟNATGAAAATTCAETATGTTGAAANO: 1465)NSNITATGGGACTATTGCTGTGATCGTH(SEQ ID NO:NOG GGOTH1A634υ 国AUGAVد[모 TELIG 80T TCTTGATTGGA 240 TRANSMEMBRANESEGMENT(aa 68-88)모JBR4ט터이 UA DP 5HHPKAVAAZαAR 100A보A민AםAAG GTGAGAGA 300P UHAR 120GCACGT 360140FAGGGACAGCACAGACTTC 420SQ 160TGAGGCTGGATCTCAA 480GSKAGCAAA180540PROTEASE-LIKEDOMAIN I(aa 121-183)SAZمTCAAAGSCGGTGY VGAATCCTGGGGGTTATGTG 660うV 200600220APICALDOMAAIN(aa 184-383)DOMAIN EXTRACELLULAR89-760) (aa1457) NO: ID SEQ ACID (AMINO1458) NO: ID SEQ (NUCLEOTIDE25WO 2025 / 155923DOMAIN EXTRACELLULAR89-760) (aa1457) ID SEQ ACID (AMINO ON1458) NO: ID SEQ (NUCLEOTIDEUMAN TRANSFERRIN RECEPTOR 1 cDNA SEQUENCE(AMINO ACID SEQ IDN 1459)HEHNUCLEOTIDE SEOGAIАстCCTGTGAATGO1PNA4Ο O>IGAA AAATGCTTTTACCLocation DOMAINSL PFN AH R터A5K 240СТАЛА 720AG 260:TCAGAGD 780GL 280τHDHτοZこん τοГАATATGAAGGAGACFH HてilTGTTG 840GH 300AGGACAT 900AQ 320PE>EHZZτοHワCAATCACACTCAG-CAGACA:SATCTOPEHHHKCAzפAGAGСTDCAGACATGT9601140RA 3401020E 3601080380Locatiation221AYAA661 GCGTAATAGT241 KD721 AAAGATATTTTGAGGE261KTA781 AAAATCTCACCTTTGCA281IIYMDα841 ATATACATGGAC301AHGE901 GCTCATCTGGC321FPPSR961 TTTCCACCATCTCG341 AAEK1021 GCTGCAGAДAAAGC361STCΣM1081 TСTACATGTAGGCAD 381LE114126401H1201421 VG1261GTAGGOKCTGAAAGAGYV(SEQATAACACTATGTTGTATATGCACAGCTCTVΟ 이Aa日EA☑A4 67P9م5ז☑A5OடR A AAαACCAG441 GFPSA1321 CAGCCCAGCAGAAGCATT CTTTG42ΣMSبA2DAEKR☑ A7 EAGAAGCTGCMТСТCAGATATGGTCTTASAGDFGلهGA ATCGGTBDCATAGTGCTGGAGACTTTGGATCG CCAGTTGGA40012004201260440 PROTEASE-LIKEDOMAINII(aa 384-605)13204601380KAFT 480 461V GA1381 GTTTGGTGCCACTGHAAGAG EGCATTTAAAGGCTTTCACT 1440り481YNILΠKA7HZKVSASP 5001441 TATATТААТСTGGATAAAGCGGTTCTTGGTACCAGCAACTTCAAGGTTTCTGCCAGCCCA 1500PCT / US2025 / 012207 Location 501 1501 521 1561 LL TGTA CTGTT FLFHHUMAN TRANSFERRIN RECEPTOR OR 1 cDNA SEQUENCE SESCAc TATATO TTICTAAFTFTTTCCCTTDYUAССT TTCPACAGATTATCСTTI[] 조CAGCA54116215611681581 PE TN1741601 LTDV1801 СТААСCCATGATGTIKCCTGAGTTGAACANA1459) (AMINO ACID SEQ ID NO: 14ACNUCLEOTIDE SEO IDTARHGGAGMCAATGΟECAGCAAAGGAATO&AMHAG ANO이Ο RA Z5こりυα 미 σ A aF ZQAAATTCTGCTEKАATGCTGAGAAAYFJGCAA天AДCTTOטDACAGATTTGTC3PYVSСTACGTATLocation DOMAINSP I E5UGIZO GAG ACFΩGGCAAACAATGСT ACECDGAGGACRIAGAGGATTIKAGTTCGTGATTAAA52015605401620560168058017406001800Hτο Π7H이HUEH RHם F H H στοU அυΠH EHI[ H こり 5 Aυ Σ AGEHP R AFH4AHG REA5 M BH A5B> GHH A이CaحرAD TCCGטaSTTTCAΩCAGFGATTTO47HRTGAGAGACATGTC6201860640192066019806802040700621 FVRD1861 TTTGTGAGGG641 WL YS1921 TGGCTGTA661 GN981681VE2041 GTGGAGTATCACTTTCAa701G21002101 TTCTGGGGCTCCGGCT720TATG7212160ΩNNGANETFRAAT216MСААААТAAD740741T2220IΟGAA21761 (SEQ ID NO:АCТАТTCAGGGAGCTGCAAATGCCCTCTCTGGTGACGTTTGGGACATTGAALSG D VWDIDNEF 760CAATGAGTTT 22801471)2281 TAA761(SEO ID NO: 14 72) 2283ATCTCACACGCTGCCAGCTT,KRKGAGAACTTGAAACTGCGTAAACGGTGCTTTTAATGAAACGCTGTTCAGAAACCAGTTGGCTCTAGCTACTTGGNHELICALDOMAIN(aa 606-760)DOMAIN EXTRACELLULAR89-760) (aa1457) NO: ID SEQ ACID (AMINO1458) NO: ID SEQ (NUCLEOTIDE27Engineered Viral Capsids

[0094] Described herein are various embodiments of engineered viral capsids, such as adenoassociated virus (AAV) capsids, that can be engineered to confer cell-selective tropism, such asCNS tissue- and cell-specific tropism, to an engineered viral particle. Engineered viral capsids canbe adenoviral or AAV capsids. The engineered capsids can be included in an engineered virusparticle (e.g., an engineered adenoviral or AAV virus particle), and can confer cell-selectivetropism to the engineered viral particle. The engineered viral capsids described herein can includeone or more engineered viral capsid proteins described herein. The engineered viral capsidsdescribed herein can include one or more engineered viral capsid proteins described herein thatcan contain one or more transferrin receptor binding modification sequence as described elsewhereherein.

[0095] The engineered viral capsids can be variants of a wild-type viral capsid. For example,in an embodiment, the engineered AAV capsids can be variants of wild-type AAV capsids. In anembodiment, the wild-type AAV capsids can be composed of VP1, VP2, VP3 capsid proteins ora combination thereof. In other words, the engineered AAV capsids can include one or morevariants of a wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins.

[0096] In an embodiment, the serotype of the reference wild-type AAV capsid can be AAV9,AAV9 K449R (or K449R AAV9), AAV1, AAVrhlO, AAV-DJ, AAV-DJ8, AAV5, AAV-PHР.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.BEST, AAV-PHP.B-GGT, AAV-PHP.B-ATP, AAV-PHP.B-ATT-T, AAV-PHP.B-DGT-T, AAVPHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.BSNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B- EGS, AAVPHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.B-DST, AAV-PHP.B-STP,AAV-PHP.B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP, AAV-PHP.B- TMP, AAV-PHP.BTTP, AAV-PHP.S / G2A12, AA VG2A 15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5(G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6,AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16,AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV 10, AAV11,AAV 12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42- lb, AAV42-2, AAV42-3a,AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11,28AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21,AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2,AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVI-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62,AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.5 1, AAV3. l / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52,AAV3-1 1l / rh.53, AAV4- 8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.1O, AAVI6. 12 / hu.l 1, AAV29.3 / bb.1, AAV29.5 / bb.2,AAV106.1 / hu.37, AAV1 14.3 / hu.40, AAVI27.2 / hu.41, AAV|27.5 / hu.42, AAVI28.3 / hu.44,AAV130.4 / hu.48, AAV145. 1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60,AAV161.6 / hu.61, AAV33.12 / hu. 17, AAV33.4 / hu.15, AAV33.8 / hu. 16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2,AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. 1,AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69,AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG- 10 / rh.40,AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5RI, AAVcy.2,AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5RI, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4,AAVcy.6, AAVhu. 1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9,AAVhu.10, AAVhu.11, AAVhu.13, AAVhu. 15, AAVhu.16, AAVhu.l 7, AAVhu.18, AAVhu.20,AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28,AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37,AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44RI,AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48RI,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55,AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66,AAVhu.67, AAVhu. 14 / 9, AAVhu .t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.1O,AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20,AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33,AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40,AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48. L2, AAVrh.48.2, AAVrh.49, AAVA.51,AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVA.61, AAVrh.64,AAVA.64R1, AAVA.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVA8R, AAVA8R A586Rmutant, AAVA8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl. 1,29AAVhErl .5, AAVhERI. 14, AAVhErl.8, AAVhErl. 16, AAVhErl. 18, AAVhErl .35, AAVhErl.7, AAVhErl .36, AAVhEr2.29, AAVhEr2.4, AAVhEr2. 16, AAVhEr2.30, AAVhEr2.3 1,AAVhEr2.36, AAVhERI .23, AAVhEr3. 1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02,AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09,AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16,AAV- LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PАEC6, АAVPAEC7, AAV-PAEC8, AAV-PAЕС 11, АAV-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, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100- 2,AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV,BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu. 19, AAVhu. 11, AAVhu.53,AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAVI28. l / hu.43, true type AAV(ttAAV), UPEN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7. 1, AAV CBr-7. 10, AAVCBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBrB7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5,AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-l, AAV CHt-2, AAV CHt3, AAV CHt-6. 1, AAV CHt-6. 10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8,AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAVCKd-1, AAV CKd-I0, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAVCKd-8, AAV CKd-B 1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKdB6, AAV CKd-B7, AAV CKd-B8, AAV CKd-HI, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4,AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl,AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAVCLg-F8, AAV CLv-l, AAV CLvl-1, AAV Clvl-1O, AAV CLvl-2, AAV CLv-12, AAV CLvl-3,AAV CLv-l 3, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv- 2, AAV CLv3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-DI, AAV CLv-D2, AAV CLv-D3, AAVCLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLvKI, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-MI,AAV CLv-MI 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8,AAV CLv-M9, AAV CLv-RI, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV30CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-IO, AAV CSp-l1,AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8. 10,AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAVCSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1,AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15,AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4,AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 andvariants thereof. See e.g., US20210380969, the content of which is incorporated by referenceherein in its entirety. In an embodiment, the serotype of the wild-type AAV capsid can be AAV9.The engineered AAV capsids can have a different tropism than that of the reference wild-typeAAV capsid.of

[0097] In an embodiment, the transferrin receptor binding modifications comprisesmodifying the AAV capsid polypeptide. In an embodiment, an engineered adeno associated virus(AAV) capsid polypeptide comprising a transferrin receptor binding modification defined by theformula X₁-X2-X3-[7-mer]-X4-Xs-X6-X7, wherein X1, X2, X3, X4, X5, X6, X7 indicatemodifications at one or more amino acid positions in the capsid polypeptide flanking the inserted7-mer (that is at each of X1, X2, X3, X4, Xs, X6, X7, the amino acid may be the wild type aminoacid at that position or an amino acid substitutions).

[0098] The core of each wild-type AAV viral protein contains an eight-stranded beta-barrelmotif (betaB to betal) and an alpha-helix (alphaA) that are conserved in autonomous parvoviruscapsids (see e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structural variable regions(VRs), also referred to as "loops", occur in the surface loops that connect the beta-strands, whichcluster to produce local variations in the capsid surface. AAVs have 12 variable regions (alsoreferred to as hypervariable regions) (see e.g., Weitzman and Linden. 2011. "Adeno-AssociatedVirus Biology." In Snyder, R.O., Moullier, P. (eds.) Totowa, NJ: Humana Press). In anembodiment, X1, X2, X3, X4, Xs, X6, and X7 modify amino acids in one or more of the 12 variableregions in the wild-type AVV capsid proteins. In an embodiment, the X1, X2, X3, X4, Xs, X6, andX7 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. In an example embodiment, the transferrinreceptor binding modification is inserted or substituted in loop IV and / or loop VIII.3119900[ In an embodiment, the transferrin receptor binding modification comprises of aminoacids 586-588 and 589-592 of a capsid protein of AAV9 (including insertion of the n-mer, such asa 7-mer, between positions 588-589), or in an analogous position of a capsid protein from AAV9,AAV9 K449R (or K449R AAV9), AAV1, AAVrhlO, AAV-DJ, AAV-DJ8, AAV5, AAV-PHP.B(PHP.B), AAV-PHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35,AAV-PHP.B2 (PHP.B2), AAV-PHP.B3 (PHP.B3), AAV-PHP.N / PHP.B-DGT, AAV-PHPР.ВEST, AAV-PHP.B-GGT, AAV-PHP.B-ATP, AAV-PHP.B-ATT-T, AAV-PHP.B-DGT-T, AAVPHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.BSNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B- EGS, AAVPHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.B-DST, AAV-PHP.B-STP,AAV-PHP.B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP, AAV-PHP.B- TMP, AAV-PHP.BTTP, AAV-PHP.S / G2A12, AA VG2A 15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5(G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6,AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16,AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV 10, AAV11,AAV 12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42- lb, AAV42-2, AAV42-3a,AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11,AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21,AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2,AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVI-7 / rh.48, AAVI-8 / rh.49, AAV2-15 / rh.62,AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.5 1, AAV3. 1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52,AAV3-1 1l / rh.53, AAV4-8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.1O, AAV16. 12 / hu.l 1, AAV29.3 / bb.1, AAV29.5 / bb.2,AAV106.1 / hu.37, AAV1 14.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44,AAV130.4 / hu.48, AAV145. l / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60,AAV161.6 / hu.61, AAV33.12 / hu. 17, AAV33.4 / hu.15, AAV33.8 / hu. 16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2,AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. 1,AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69,AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG- 10 / rh.40,AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5RI, AAVcy.2,32AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5RI, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4,AAVcy.6, AAVhu. 1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9,AAVhu. 10, AAVhu. 11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.l 7, AAVhu.18, AAVhu.20,AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28,AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37,AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44RI,AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48RI,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55,AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.6l, AAVhu.63, AAVhu.64, AAVhu.66,AAVhu.67, AAVhu.14 / 9, AAVhu .t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.1O,AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20,AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33,AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40,AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48. L2, AAVrh.48.2, AAVrh.49, AAVA.51,AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVA.61, AAVrh.64,AAVA.64R1, AAVA.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVA8R, AAVA8R A586Rmutant, AAVA8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl. 1,AAVhErl.5, AAVhERI. 14, AAVhErl .8, AAVhErl. 16, AAVhErl. 18, AAVhErl.35, AAVhErl.7, AAVhErl .36, AAVhEr2.29, AAVhEr2.4, AAVhEr2. 16, AAVhEr2.30, AAVhEr2.3 1,AAVhEr2.36, AAVhERI .23, AAVhEr3. 1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02,AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09,AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16,AAV- LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAVPAEC7, AAV-PAEC8, AAV-PAEC 11, AAV-PAEC 12, AAV-2-pre-miRNA-I01, AAV-8h,AAV- 8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2,AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV,BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu. 19, AAVhu. 11, AAVhu.53,AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128. I / hu.43, true type AAV(ttAAV), UPEN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7. 1, AAV CBr-7. I0, AAV33CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBrB7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5,AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt3, AAV CHt-6. 1, AAV CHt-6. 10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8,AAV CHt-PI, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAVCKd-1, AAV CKd-IO, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAVCKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKdB6, AAV CKd-B7, AAV CKd-B8, AAV CKd-HI, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4,AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl,AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAVCLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3,AAV CLv-l 3, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv- 2, AAV CLv3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-DI, AAV CLv-D2, AAV CLv-D3, AAVCLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLvKI, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-MI,AAV CLv-MI 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-М8,AAV CLv-M9, AAV CLv-RI, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAVCLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-1O, AAV CSp-1 1,AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8. 10,AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAVCSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1,AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15,AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4,AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 andvariants thereof. In an embodiment, X1-X2-X3 comprises of any sequential amino acids 449-459and X4-X5-X6-X7 comprises of any amino sequential amino acids 452-463 of a capsid protein ofAAV9, or in an analogous position of a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAVrh.10.

[0100] In an embodiment, the transferrin receptor binding modification is incorporated into aviral protein, such as a capsid protein, including but not limited to adenoviral or AAV proteins. Inan embodiment, the n-mer is located between two amino acids of the viral protein such that the34transferrin receptor binding modification is external (i.e., is presented on the surface of) to a viralcapsid. In an example embodiment, the n-mer disclosed herein can be inserted between twoconsecutive amino acids in the wild-type viral protein (VP) (or capsid protein), including in regionsthat are surface exposed when incorporated into a viral capsid. In an embodiment, the n-mer canbe inserted between two consecutive amino acids in a variable amino acid region in a viral capsidprotein.

[0101] In an embodiment, the n-mer can be inserted between two consecutive amino acids ina variable amino acid region in an AAV capsid protein. In an embodiment, one or more n-mer canbe inserted between two amino acids in one or more of the 12 variable regions in the wild-typeAVV capsid proteins. In an embodiment, the one or more n-mers can be each be inserted betweentwo amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VI, VR-III, VR-IX, VR-X,VR-XI, VR-XII, or a combination thereof. In an example embodiment, the transferrin receptorbinding modification is inserted or substituted in loop IV and / or loop VIII. In an exampleembodiment, the n-mer or 7-mer is YSRIGPN (SEQ ID NO: 1390), YSRNSDN (SEQ ID NO:1391), LHRLGPN (SEQ ID NO: 1392), FRSTNGV (SEQ ID NO: 1393), FVSTNGV (SEQ IDNO: 1394), FZ1STNGZ2 (SEQ ID NO: 1395), FRSTNGZ3 (SEQ ID NO: 1396), and VESTNGR(SEQ ID NO: 1397). In an embodiment, Z1 is selected from the group consisting of A, D, H, N,Q, and S; Z2 is selected from the group consisting of K and R; and Z3 is selected from the groupconsisting of L, M, and R.

[0102] In an embodiment, the engineered capsid is a modified AAV1 capsid and can have a nmer motif inserted after or a neighbor of amino acid 590 (i.e., between amino acid 590 and 591).In an embodiment, the engineered capsid is a modified AAV3 capsid and can have a n-mer motifinserted after or a neighbor of amino acid 586. In an embodiment, the engineered capsid is amodified AAV4 capsid and can have a n-mer motif inserted after ora neighbor of amino acid 586.In an embodiment, the engineered capsid is a modified AAV5 capsid and can have a n-mer motifinserted after or a neighbor of amino acid 575. In an embodiment, the engineered capsid is amodified AAV6 capsid and can have a n-mer inserted at or a neighbor of amino acid 585 andoptionally Y705-731, T492V, K531E. In an embodiment, the engineered capsid is a modifiedAAV8 capsid and can have a n-mer inserted after or a neighbor of amino acid 585 and 590. In anembodiment, the engineered capsid is a modified AAV9 capsid and can have a n-mer inserted inbetween amino acid 588 and 589. (Büning, H.; Srivastava, A. Capsid Modifications for Targeting35and Improving the Efficacy of AAV Vectors. Molecular Therapy - Methods & ClinicalDevelopment 2019, 12, 248-265). In an embodiment, the engineered capsid can have a 7-mermotif inserted between amino acids 588 and 589 of an AAV9 viral protein. SEQ ID NO: 1 is areference AAV9 capsid sequence for at least referencing the insertion sites discussed above. In anembodiment, the engineered capsid can have a 7-mer motif inserted between two consecutiveamino acids within amino acids 451-460 of a capsid protein of AAV9 viral protein. SEQ ID NO:1 is a reference AAV9 capsid sequence for at least referencing the insertion sites discussed above.

[0103] It will be appreciated that n-mers can be inserted in analogous positions in AAV viralproteins 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, AAVPHP.B (PHP.B), AAV-PHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32,AAVTH1.1-35, AAV-PHP.B2 (PHP.B2), AAV-PHP.B3 (PHP.B3), AAV-PHP.N / PHP.B-DGT,AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.B-ATP, AAV-PHP.B-ATT-T, AAV-PHP.BDGT-T, AAV-PHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAVPHP.B-SNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B- EGS,AAV-PHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.B-DST, AAV-PHP.BSTP, AAV-PHP.B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP, AAV-PHP.B- TMP, AAVPHP.B-TTP, AAV-PHP.S / G2A12, AA VG2A 15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5(G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6,AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16,AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV 10, AAV11,AAV 12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42- lb, AAV42-2, AAV42-3a,AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11,AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21,AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2,AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVI-7 / rh.48, AAVI-8 / rh.49, AAV2-15 / rh.62,AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.5 1, AAV3. 1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52,AAV3-1 1l / rh.53, AAV4-8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.1O, AAV16. 12 / hu.l 1, AAV29.3 / bb.1, AAV29.5 / bb.2,AAV106.1 / hu.37, AAV1 14.3 / hu.40, AAVI27.2 / hu.41, AAV127.5 / hu.42, AAVI28.3 / hu.44,AAV130.4 / hu.48, AAV145. 1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60,36AAV161.6 / hu.61, AAV33.12 / hu. 17, AAV33.4 / hu.15, AAV33.8 / hu. 16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2,AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. 1,AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69,AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG- 10 / rh.40,AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5RI, AAVcy.2,AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5RI, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4,AAVcy.6, AAVhu. 1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9,AAVhu. 10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.l 7, AAVhu.18, AAVhu.20,AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28,AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37,AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44RI,AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48RI,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55,AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66,AAVhu.67, AAVhu. 14 / 9, AAVhu .t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.1O,AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20,AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33,AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40,AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48. L2, AAVrh.48.2, AAVrh.49, AAVA.51,AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVA.61, AAVrh.64,AAVA.64R1, AAVA.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVA8R, AAVA8R A586Rmutant, AAVA8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl. 1,AAVhErl .5, AAVhERI. 14, AAVhErl.8, AAVhErl. 16, AAVhErl. 18, AAVhErl .35, AAVhErl.7, AAVhErl .36, AAVhEr2.29, AAVhEr2.4, AAVhEr2. 16, AAVhEr2.30, AAVhEr2.3 1,AAVhEr2.36, AAVhERI .23, AAVhEr3. 1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02,AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09,AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16,AAV- LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAVPAEC7, 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, AAV37Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100- 2,AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV,BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu. 19, AAVhu. 11, AAVhu.53,AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAVI128. I / hu.43, true type AAV(ttAAV), UPEN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7. 1, AAV CBr-7. 10, AAVCBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBrB7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5,AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-l, AAV CHt-2, AAV CHt3, AAV CHt-6. 1, AAV CHt-6. 10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8,AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAVCKd-1, AAV CKd-IO, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAVCKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKdB6, AAV CKd-B7, AAV CKd-B8, AAV CKd-HI, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4,AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl,AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAVCLg-F8, AAV CLv-l, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3,AAV CLv-13, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv- 2, AAV CLv3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-DI, AAV CLv-D2, AAV CLv-D3, AAVCLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLvKI, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-MI,AAV CLV-MI 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8,AAV CLv-M9, AAV CLv-RI, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAVCLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-IO, AAV CSp-1 1,AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8. 10,AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAVCSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1,AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15,AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4,AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 andvariants thereof. See e.g., US20210380969, the content of which is incorporated by reference38herein in its entirety. In an embodiment as previously discussed, the n-mer(s) can be insertedbetween any two contiguous amino acids within the AAV viral protein and in an embodiment theinsertion is made in a variable region.

[0104] In an embodiment, the first 1, 2, 3, or 4 amino acids of a transferrin receptor bindingmodification can replace 1, 2, 3, or 4 amino acids of a polypeptide into which it is inserted andpreceding the insertion site. Using an AAV as another non-limiting example, one or more of then-mers can be inserted into e.g., an AAV9 capsid polypeptide between amino acids 588 and 589and the insert can replace amino acids 586, 587, and 588 such that the amino acid immediatelypreceding the transferrin receptor binding modification after insertion is residue 585. It will beappreciated that this principle can apply in any other insertion context and is not necessarily limitedto insertion between residues 588 and 589 of an AAV9 capsid or equivalent position in anotherAAV capsid. In an example embodiment, the AAV capsid protein is selected from SEQ ID NO:1.39Table B. Example AAV VP1 amino acid sequencesSerotype SEQ ID NO: Accession numberAAV13 NP 049542.1AAV2 YP 680426.14AAV35 NP 043941.1AAV3B6 3KIC AVP1 AMINO ACID SEQUENCEMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDEPVNAADAAALEHDKAYDOOLKAGDNPYLRYNHADAEFOERLOEDTSFGGNLGRARAVFOAKKRVLEPLGLVEEGAKTAPGKKRPVEOSPOF.PDSSSGIGKTGOOPAKKRLNFGOTGD IGDSESVPDPO CEEPAPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVRVITTSTRTWA LPTYNNHLYKOISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI ORIINNNWGFRPK RLNFKLFNIOVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCL GCLPPFPADVEMIPOYGYLTLNNGSOAVGRSSFYCLEYFPSOMLRTGNNFTFSYTFEEVPFHSISSYAHSOSLDRLMNPLIDQYLYYLNRTONOSGSAONKDLLFSRGS PAGMSVOPKNWLPGPCY YROORVSKTKTDN MNPLIDQYLYYLNRIONOSGSAQNKDELESRGSPAGMS VOPRNWLPGPCY IRØQRVSRIRIDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKEKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDRDVYLOGPIWA KIPHTDGHFHPSPLMGGFGLKNPPPOILIKNTPVPANPPAEFSATKFASFITOY TQYSTGQVSVE TEWELOKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGLGPFNGLDKG EPVNEADAAALEHDKAYDROLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGLGRAVFOAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGOOPARKRLNINEGOTGDADSVPDPQPLGOPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHСCDSTWMGDRVITTSTRTWALPTYNNHLYKOISSOSGASNDNHYFGYSTPWGYFDENREHCHHFSPRDWORLINNNWGFRPKR енэRDWQRINNNWGIRERRLNFKLFNIQVKEVTONDGTTTIANNLTSTVOVFTDSEYOLLPYVLGSAHOGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNETF'S.sYTFEDVPFHSSYAHSQSLDRLM NPLIDOYLYYLSRTNTPSGTTTOSRLOFSOAGASDIRDOSSRNWLPGPCYRQQRVSKTSADNN NSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQOSGVLIFGKOGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLORGNRQAATADVNTOGVI OGVLPGMVWODRDVYLOGPIWAK 1PHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTESAAKFAS AAKFASFITOYSTGOVSVEI EWELOKENSKRWNPEIOYTSNYNKSUNVDETVDTNGVYSEPRPIGTRY1 RYLTRNLMAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGGYKYLGPGNGLDKG NLSEGIREWWALREGVEOHADOERLOEDTS EPVNEADNDNARLENDRĄ ADAAALEHDKOKAYDOOLKAGDNPYLKYNHADAEFOOERLOEDTSFGGNLGRAVFOAKKR BE VNE FQERLQEDTSEGGNLGRAVEQAKKRLEPLGLVEEААКТАPGKKG PGKKGAVDOSPOEPDSSSGV SGVGRSGKQPARKRLNFGQTGDSESVPDPQ GVGKSGKOPARKRLNFGOTGDSESVPDPQPLGEPPAAPTSLGSNTMASGG EGADGLPTYNNHLYKOISSOSGASNDIONHYFGYSTPWGYGYFDFNRFHCHFSPRDWORLINNNWGFRPKKLSFKLFNIOVRGVTONDGTTT\'IANNLTSTVOVE"VFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPOYGYLTLNNGSOAVGRSSFYCLEYFPSOMLRTGNNFOFSYTFEDVPFHSSYAHSOSLDRLMNPLIDQYLYYLNRTOGTTSGTTNOSRLLFSOAGPOSMSLOARNWLPGPCYROORLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLOSSNTAPTTGTVNHOGALPGMVWODRDVYLOGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGOVSVEIEWWELOKENSKRWNPEIOYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNLLPDWLEDNLSEGIREWWALKPGVPQPKANQOHODNRRGLVLPGYKYLGPGNGLDKGEPVNFADAAALEHDKAYDOOLKAGDNPYLKYNHADAEFO HADAEFQERLQEDTSFGGNLGRAVFQAKKR40Serotype SEQ ID NO: Accession numberAAV47 NP 044927.1AAV5AAV68のYP 068409.1AAB95450.1VP1 AMINO ACID SEQUENCEILEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGOTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSOWLGDRVITTSTRTWALPTYNNHLYKOISSOSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNNNWGFRPKKLSFKLFNIQVKEVTONDGTTTIANNLTSTVOVFTDSEYOLPYVLGSAHOGCLPHРРЕРADVFMV LSFRLENIQVREVTONDGTTTIANNLISTVOVETDSEYQLPIVLGSAHQGCLPEPOYGYLTLNNGSQAVGRSSFYCLEYFPSOMLRTGNNFOFSYTFEDVPFHSSYAHAHSOSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLS RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNV MITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLOGPIWA KTPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVE IEWELOKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL MTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKG MIDGYLPDWLEDNLSEGVLEVNHADAEFOORLOGDTSFGGNLGRAV KGE PVNAADAAALEHDKAYDOOLKAGDNPYLKYNHADAEFQQRLOGDTSFGGNLGRAVEVFOAKKRVE VNAADAAAERDAIDRASDNE IENINADASIYOISANTO LEPLGLVEQAGETAPGKKRPLIESPOOPDSSTGIGKKGKQPAKKKLVFEDETGAGAGDGPPEGS VEQAGET. SAGDGEPEGSSDDSEMR RTWVLPTYNNKAMRVKIFNIQ HLYKRLGESLQSNTYNGFSTPWGYFDFNRFHCHFSPRDWORLINNNWGMRPKA VKEVTTSNGETTVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPNDVFMV TGNTSOOOTDRNAFYCLEYFPSOMLRTGNNFEITYSFEKVPFHSMYAHSOSLMVPOYGYCGLV HSOSLDRLMNPLIDOTANONYKIPATENTATVPGTLIFYLWGLOSTTTGTTLNAGTATTNFTKLRPTNFSNFKKNWLPGPSIKOOGFSKTTIDGRWSALTPGPPMATAGPADSKESNSOLIFAGPKONGM DSLIKYETHSTI GSDSLEEELAATNATDT TSE OTDMWGNLPGGDOSNSNLPTVDRLTALGAVТDGHFHPSPLIG PHTIVPGMVWONRDIYYOGPIWAKIFSSTPVNSFITQYSTGQVSVQID GGFGLKHPPPQIFIKNTPVPANPATTFRSKRWNPE WEIOKERS LTHHL PEVOFTSNYGOONSLLWAPDAAGKYTEPRAIGTRYLNYLGPGNGLDRGE MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNOOHODOARGLVLPGYNPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNGGNLGKAVFQAKKRVEGAKT EAGPSGSOOLOIPAQPASSLGADTOTMSAGGGGPPLGDNNOGADGVGNASGDWHCDSTWMGDRVVVTKSTRTWVLPSYNNHOYR NAYFGYSTPWGYFDFNRFHSHWSPRDWORL EIKSISGSVDGSNANA RLINNYWGFRPRSLRVKIFNIQ ANNLTSTVQVFTDDDYQLPYVVGNGTEG VKEV CEGCLPAFPPOVFTLPOYGYATLN DNTENPTERSSEFCLEXE NN FPSKML.RTGNNFEFTYNFEEVPPEHSSFAPSONLEKLANPLVDOY LYRFVSTNNTGGVOFNKNLAGRYANTYKNWFPGPMGRTO GWNLGSGVNRASVSAFATTNRMEEGASYOVPPOPNGMTNNLOGOGSNTYALENTMIFNSOPA OPANPGTTATYLEGNMLITSESETOPVNRVAVAYNVGGOMATNNOSSTTANAPATGTYNLOEIVPOPGSVWMERDVYLOGPIWAKIPETGAHFHP RVAINVSGQPAINNØ951TAESPPAMGGFGLKHPPPMMLIKNTPPVPGNITSFSDVPVPVSSFITOYSTGOVTVEMEWELKKENSKR EAMGGEGLKHPPEMMLIKNIPVEGNWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPLAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANOOKODDGRGLVLPGYKYLGPFNGLDKGEPVIVNAADAAALEHDKAYDOOLKAGDNPYLRYNHADAEFQERLOEDTSFGGNLGRAVFOAKKRVLEEPFGLVEEGAKTAPGKKRPVEOSPOEPDSSSGIGKTGOOPAKKRLNFGOTGDSESVPDPOGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWAIPTYNNHLYYKOISSASTGASNDNHYFGY WGYFDFNRFHCHFSPRDWORLINNNWGFRPK41Serotype SEQ ID NO: Accession number42WO 2025 / 155923VP1 AMINO ACID SEQUENCERLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHOGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSOSLDRLMNPLIDQYLYYLNRTONOSGSAQNKDLLFSRGS RVSKNNSNEIWIGASRININGRESIINPGIAMASHKDDADAEFEMSGVMIFGNESAGASNTALCNAYLOGPKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITOYSTGOVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGVLEPLGLVEEGAKTAPAKKRPVEPSPORSPDSSTGIGKKGQQPARKRLNFGQTGIOPLGEPPAAPSSVGSGTVAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVALPTYNNHLYKOISSETAGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWORLIVFOAKKRGDSESVPDPRVITTSTRTWLINNNWGFRPCLPPFPADVFdSYAHSQSLDK FRQQRVSKTLDFGKTGATNKTTLENKKLRFKLFNIOVKEVTTNDGVTTIANNLTSTIQVFSDSEYQLPYVLGSAHOGOMIPQYGYLTLNNGSQSVGRS NNETSTIQVFSDSEYQLPIVLGSAN EYFPSOMLRTGNNFEFSYSFEDVPFHSS LMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPSTMAEQAKNWLPGPCFRONNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFGKTDGALPGMVWONRDVYLOGPIW VLMTNEEEIRPTNPVATEEYGIVSSNLOAANTAAOTOVVNNOGALPOAKIРHTDGNFHPSPLMGGFGLKHPPPOILIKNTPVPANPPEVFTPAKFASFITOEIEWELOKENSKRWNPEJOYTSNFEKOTGVDFAVDSOGVYSEPRPIGTRYLTRNVLPGYKYLGPFNGLDKG MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVI DAAALEHDKAYD EPVNAADAVEEGAKTAP VLEPLGLVELOAKTAДДPSGVGP OPLGEPРРAA OPLGEPPAPALPTYNNYDOOLOAGDNPYLRYNHADAEFQERLOLQEDTSFGGNLGRAVFQAKKRARKRLNFGOTGDSESVPDP APGKKRPVEPSPORSPDSSTGIGKKGOOPARAARLNរDSLSVEDE ENWHCDSTWLGDRVITTSTRTW PNTMAAGGGAPMADNNEGADGVGSSSGNGNWHCDSTWLGDRVITTSTRIW PNTMAAGGGAPMADNNEBGADEVGDSSGNGTSGGATNDNTYFGYSTPWGYFDFN NNHLYKQISNGT NRFHCHFSPRDWQRLINNNWGFRDSEYQLPYVLGSAHQGCLPPFPADV EGTKTIANNLTSTIOVFTDS LSFKLFNIQVKEVTONE PKRLSSFYCLEYFPSOMLRTGNN PQYGYLTLNNGSQAVGRSS FMIPMNPLIDOYLYYLSRTOTTGO PNTMANQAKNWLPGPCYROORVSTTTG TGGTANTOTLGFSOGGPNTM SNFAWTAGTKYHLNGRNS ONNNSN DDEEREFPSNGILIFGKONAARDNADYS GRNSLANPGIAMATHKDDEEREF SEEEIKTTNPVATEEYC DVMLTSEE VNSOGAL.PGMVWONRDVYLOGPI YGIVADNLOOONTAPOIGTYGLKHPPPOILIKNTPVPADIAAV7 10 YP 077178.1AAV8 11 YP 077180.1WAKIPHTVEIEWE уаыеыNDETOVTSNVYKST VELOKENSKRWNPEIOYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNLMAДADGYLPDWLEDNLSEGIREWWALKPGAPOPKANOOHODNARGLVLPGYKYLGPGNGLDKGAAV91 AAS99264.1EPVNAADAAALEHDKAYDOOLKAGDNPYLKYNHADAEFOERLKEDTSFGGNLGRAVFOAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGOTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKOISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRPкKRLNFKLFNIOVKEVTDNNGVKTIANNLTSTVOVFTDSDYOLPYVLGSAHEGCLPPFPADVFМТPOYGYLTINDGSOAVGRSSFYCLEYFPSOMLRTGNNFOFSYEFENVPFHSSYAHSOSLDRNPLIDOYLYYLSKTINGSGONOOTLKFSVAGESNMAVOGRNYIPGPSYROORVSTTVTONPCT / US2025 / 012207Serotype SEQ ID NO: Accession number43AAV92 K449RVP1 AMINO ACID SEQUENCENNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWODRDVYLOGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITOYSTGOVSVE RIEHIDGNERESELMGGEGMANEEFQILIRNIEVEADEETAENADAENSFITQIS IEWELOKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*MAADGY ADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQ PIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWA LPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRP KRLNFKLFNIOVKEVTDNNGVKTIANNLTSTVOVFTDSDYOLPYVLGSAHEGCLPPFPADVFMIPOYGYLTLNDGSQAVGRSSFYCLEYFPSOMLRTGNNFOFSYEFENVPFHSSYAHSQSLDRWO 2025 / 155923LMNPLIDOYLYYLS RTINGSGONOOTLKFSVAGPSNMAVOGRNYIPGPSYROORVSTTVTONNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGOVATNHOSAQAQAQTGWVONQGILPGMVWQDRDVYLOGPIW VMITNEEEIKTTNEVATESIGQVATNHOSAYADAIGWVONOSILEGI АКІРНТDGNFHPSPLMGGFGMKHPPPOILIKNTPVPADPPTAFNKDKLNSFITOYSTGOVSVEIEWELOKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKDKGOTGESESVPDP EPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAK KTAPGKKRFVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGOT GLVEEAAKT. VLEPLGI TWLGDRVITTSTRTW MAAGGGAPMADNNEGADGVGSSSGNWHCDSTV GEPPAGPSGLGSGTMA OPIGEALPT FNRFHCHFSPRDWORLINNNWGFR GSTNDNTYFGYSTPWGYFDE RLSFKLENIOVKEVTONEC PKR VFTDSEYOLPYVLGSAHOGCLPPFPADV GTKTIANNLTSTIOVESSFYCLEYCL RLMNPLIDOYLYYLSRTOSTGGTOGTOOLLFSOAGPANMSAOAKNWLPGPCYROORVSTTLSONNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKOGAGRDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLOOANTGPIVGNVNSOGALPGMVWONRDVYLOGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQI NTPVPADPPTTFSQAKLASFITOYSTGOVSYTSNYYKSTNVDFAVNTEGTYSEPRE VEIEWELOKENSKRWNPEIOYTSNYYKSAAV10 15 AAT46337.1 FMAAV11 16 AAT46339.1CWWDLKPGAPKPKANOOKKODDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFOERLOEDTSFGGNLGRAVFOAKKRVLEPLGLVEEGAKTAPGKKRPLESPQEPDSSSGIGKKGKQPARKRLNFEEDTGAGDGPPEGSDTSAMSSDIEMRAAPGGNAVDAGOGSDGVGNASGDWHCDSTWSEGKVTTTSTRTWVLPTYNNHLYLRLGTTSSSNTYNGFSTPWGYFDFNRFHCHFSPRDWORLINNNWGLRPKAMRVKIFNIO HкотCCNIINGISIEWGIIDINREHeHrrRDWQRLINNNWGIREKAMRVRIINIQ VKEVTTSNGETTVANNLTSTVOIFADSSYELPYVMDAGOEGSLPPFPNDVFMVPOYGYCGIV REVI1SNGETIVANNLISIVQIFADSSIELEIVMDAGQEGSLEFEENDVEMVEQIGICGIVTGEGENONOTDRNAFYCLEYFPSOMLRTGNNFEMAYNFEKVPFHSMYAHSOSLDRLMNPLLDOYLWHLHLQSTTSGETLNQGNAATTFGKIRSGDFAFYRKNWLPGPCVKQQRFSKTASQNYKIPASGGNALLKLKYDTHYTLNNRWSNIAPGPPMATAGPSDGDFSNAQLIFPGPSVTGNTTTSANNLLFTSEEEIAАAATNPRDTDMFGOIADNNONATTAPITGNVTAMGVLPGMVWONRDIYYOGPIWAKIPPCT / US2025 / 012207Serotype SEQ ID NO: Accession number44WO 2025 / 155923VP1 AMINO ACID SEQUENCEHADGHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFTAARVDSFITQYSTGQVAVQIEWEIEKERSKRWNPEVQFTSNYGNQSSMLWAPDTTGKYTEPRVIGSRYLTNHLMAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQOHODNGRGLVLPGYKYLGPFNGLDKG MAADGYLEDWLEDNESEGIREWWALRPGAPQPRANQQHODNGRGLVLPGYRYLGEEPVNEADAAALEHDKAYDKOLEOGDNPYLKYNHADAEFOORLATDTSFGGNLGRAVFOAKKRILEPLGLVEEGVKTAPGKKRPLEKTPN TPNRPTNPDSGKAPAKKKQKDGEPADSARRTLDFEDSG AGDGPPEGSSSGEMSHDAEMRAAPGGNAVEAGQGADGVGNASGDWHCDSTWSEGRVTTTSTR TWVLPTYNNHLYLRIGTTANSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNWGLRPKS MRVKIFNIOVKEVTTSNGETTVANNLTSTVQIFADSTYELPYVMDAGQEGSFPPFPNDVEMY MRVKI ENTONONOTDRNAFYCLEYFPSQMLRTGNNFEVSYQFEKVPFHSMYAHSQSLDR MMNPLLDOYLWHLOSTTTGNSLNQGTATTTYGKITTGDFAYYRKNWLPGACIKQQKFSKNANONYKIPASGGDALLKYDTHTTLNGRWSNMAPGPPMATAGAGDSDFSNSOLIFAGPNPSGNTTTSSNNLLFTSEEEIATTNPRDTDMFGOIADNNONATTAPHIANLDAMGIVPGMVWONRDIYYOGPIWAKVPHTDGHFHPSPLMGGFGLKHPPPQIFIKNTPVPANPNTTESAARINSFLTQYSTGQVAVQIDWEIQKEHSKRWNPEVQFTSNYGTQNSMLWAPDNAGNYHELRAIGSRFLTHHLMTDGYLPDWLEDNLSEGVREWWALOPGAPKPKANOOHODNARGLVLPGYKYLGPGNGLDKGE MIDGYLEDLEAYDQOLKAGDNPYLKYNHADAEFQERIQEDTSFGONLORAO QAKKRI DTESVPDPOP LEFLGLVEEAAKTAPGKKRPVEQSPAEPDSSSGIGKSGQQPARKRLNFGQTGDT WHCDSOWLGDRVITTSTRTWAL IGOPPAAPSGVGSTTMASGGGAPMADNNEGADGVGNSSGNWPTYNN HCHFSPRDWORLINNNWGFRPKRL IHYFGYSTPWGYFDFNRFHYGYLTLNNGSOAVGRSSFYCLEYFPSOMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNAAV12 17 ABI16639.1NFKLFNIOVKEVTONDGTTTIANNLTS AAV13 18 ABZ10812.1PLIDQYLYYLNRTOTASGTOOSRLLFSOAGPTSMSLOAKNWLPGPCYROORLSKOANDNNNS DLIIINKIQTASGIQQSRELESQAGETSMƏLQAKNWLEGFCIRQQRESKQANDNNNSNFP NEPEWTGATKIHLNGRDSLVNPGPAMASHKDDREЕкFFPMHGILIIGKEGINANNADLENVMITDEEEIRTTNPVATEQYGTVSNNNLONSNAGPTTGGTVNHQGALPGMVWQDRDVYLQGPIWAKIPTDGHFHPSPLMGGFGLKHPPPOIMIKNTPVPANPPTNAAVrh.8ELOKENSKRWNPEIOYTSNYNKANPPTNFSAAKFASFITOYSTGOVSVEIEWGVYSEPRPIGTRYLTRNL WKSVNVDFTVDTNGVYSEPMAADGYLPDWLEDNLSEGIREW KODDGRGLVLPGYKYLGPFNGLDKG REWWDLKPGAPΚΡKΑNΩΟΚΟΙEPVNAADAAALEHDKAYDQQLKAKAGDNPYLRYNHADAEFQE FOERLOEDTSFGGNLGRAVFOAKKR VLEPLGLVEEGAKTAPGKKRPVEQSPQOEPDSSSGIGIGKTGOOPAKKRLNFGOTGDSESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWA STNTMASGGGAPMADNNLPTYNNHLYKOISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRPKRLNFKLFNIOVKEVTTNEGTKTIANNLTSTVOVFTDSEYOLPYVLGSAHOGCLPPFPADVF KRLNEKLENIQVKEVTTNEGIKTTANNLTSTVQVETDSEYQLPYVLGSAHQGCLPPFPADVEMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNQNNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKQGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGOVSVEIEWELOKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL12 AA088183.1PCT / US2025 / 012207WO 2025 / 155923Serotype SEQ ID NO: Accession numberAAVrh. 13 10 AA088201.1VP1 AMINO ACID SEQUENCEMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANOOKODDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFOAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPORSPDSSTGIGKKGQQPAKKRLNFGOTGDSESVPDPOPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTW QPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADEVESSSGNWHCDSIWEGDRVITISIRIWALPTYNNHLYKOISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRPKRLNFKLFNIQVKEVTONEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADV FMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFEDVPFHSSYAHSQSLD RLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLS ONNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYS SVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPI WAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVS WAKIPHTDGNYTSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL VEIEWELOKENSKRWNPEIOYTSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANOOKODNGRGLVLPGYKYLGPFNGLDKG MAADGYLEDWLEDNLSEGIREWWDLRPGAPRPRANOOKODNGRGLVLEGIAIEGPENGEDRGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKR VLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDP QPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV45PCT / US2025 / 012207ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSFSPRDWORL SEO ID NO: 2 AAVrh7 14 of4 US20220370639INNNWGFRPKRLNFKLFNIQVKEVTONEGTKTIANNLTSTIQVFTDSEYQLPYVIVLGSAHOGC LPPFPADVEMIPOYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNENFEDVPFHSSYAHSOSLDHDRLMNPLPLIDOYLYYLSRTOSTGGTAGTOOLLFSOAGPNNMSAOAKNWLPGPCYHROORVSTTTTLSONNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDEERFFPSSGVLMFGKOGOGAGKDNVDDYSSVMLTSEEEIKTTNPVATEOYGVVADNLOOONAAPIVIVGAVNSOGALPGMVWONONRDVYLOG RNRDVIIQGGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPAPADPPTTFNQ AKLASFITITQYST.GOGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRE LTRNLAAV- 19 AGT20780.1 LK19AAVAV-DJ 20 3J1Q ARPIGTRYLMAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPOEPGNGLDKG bAAALEHDKAYDOOLKAGDNPYLKYNHADAEFOERLKEDTSFGGNLGRA GRAVFOAKKRLLEPLGLLVEFAAKTAР 2EPDSS5GVONSSGNWHCDSQUWLGD SGVGKSGKQPARKRLNFGOTGTGDSESVPDPO PLGEEPPAAPTSLGSNTMASGGG SDRVITTSTRTWAT.PmTYNNHLYKOISSOSGASAASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLRLINNNWGFRPKK SISFKLFNIOVKEVTONDGTTT uoCCI TTIANNLTSTVOVFTDSEYOLPYVLGSAHOGCECLPPFPADVFMVOYGYLTLNNGSOAVGRSSFYYCLEYFPSOMLRTGNNFOFSYTFEDVPFHSSsSYAHSOSLDRLMPLIDOYLYYLNRTOGTTSGT NITIDNRIQGISG.TTNOSRLLFSOAGPOSMSLOARNWLPGPCYROC TINOSRLEE SQAGPOSMSLQARNWLPGPCIROL ORLSATANDN NNSNFPWTAASKYHLNGRDSLVVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTАTASNAELDNVMITDEEEIRTTNPVATEQYGTVTVANNLOSSNTAPTTRTVNDOGALPGMVWODRDVDVYLOGPIWAKIPHTDGHFHPSPLMGGFGLKHKHPPPOIMIKNTPVPANPPTTFSPAKFASFITOYS OYSTGOVSVEEWELOKENSKRWNPEIOYTSA TSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPMAADGYIYLPDWLEDTLSEGI SEGIROWWKLKPGPPPPKPAERHKDDSRSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDROLDSGDNPYLKYNHADAEFOERI.KEDTSFGGNLGRGRAVFOAKKRSerotype SEQ ID NO: Accession number46VP1 AMINO ACID SEQUENCELLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGOOPARKRLNFGOTGDADSVPDPOフクロウブヒロ PIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRV KRLSFKLFNIMIPOYGYIRVITTSTRTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWORI CHESPRDWORIINNNWOINE RLINNNWGFRPNIQVKEVTQNONEGTKTIANNLTSTIOVFTDSEYOOLPYVLGSAHOGCLPPFPADVE NEGIRIIANNLTSTTQVETDSEYMIPQYGYTYLTLNNGSQAVGVGRSSFYCLEYFPSOMLRTGNNFOFTYTFEDVPFHSSYAHSOSLDRLMNPLIDQYI DOYLYYLSRTOT TOTTGGTTNTQTLGFSQGGPNTMANNQAKNWLPGPCYRQQRVSKTSADTGATKYHL SGVLIFGKQGSEKTNVDIVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTOGVLPGNAKIPHTDGHFHPSPLMGGFGLKHPPPOILIKNTPVPADPPTTFNOSKLNSFITEIEWELOKENSKRWNPEIOYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNIPGMVWODRDVYLOGKINSFITOYSTGOVSV

[0105] In an embodiment, the 7-mer comprises YSRIGPN (SEQ ID NO: 1390), YSRNSDN(SEQ ID NO: 1391), or LHRLGPN (SEQ ID NO: 1392), and wherein X₁ comprises A, G, E, L,N, Q, S, W, or M; X2 comprises A, F, I, L, M, N, Q, P, T, V, or Y; X3 comprises Q; X4 comprisesany amino acid; Xs comprises D, F, G, I, L, M, Q, P, S, T, or V; X6 comprises A, C, F, G, H, I, Р,S, T, V, W, or Y; X, comprises D, E, Q, or T; or any combination thereof. In an embodiment, thetransferrin receptor binding modification comprises of the amino acid sequence of one of SEQ IDNos: 60-1210.

[0106] In an embodiment, the n-mer comprises of YSRIGPN (SEQ ID NO: 1390), and whereinX₁ comprises of A, G, E, L, N, Q, S, W, or M;X2 comprises of A, F, I, L, M, N, Q, P, T, V, or Y;X3 comprises of Q; X4 comprises of A, E, F, H, I, L, M, N, P, Q, V, Y, D, or G; Xs comprises ofD, F, G, I, L, M, Q, P, S, T, or V; X6 comprises of A, C, F, G, H, I, P, S, T, V, W, or Y; X7comprises of D, E, Q, or T; or any combination thereof. In an embodiment, the transferrin receptorbinding modification comprises of the amino acid sequence of one of SEQ ID Nos: 60-1136.

[0107] In an embodiment, the n-mer comprises of YSRNSDN (SEQ ID NO: 1391) and X1comprises of S, or W; X2 comprises of V, I, or F; X3 comprises of Q; X4 comprises of any aminoacid; Xs comprises of Q or T; X6 comprises of A; X7 comprises of Q; or any combination thereof.In an embodiment, the transferrin receptor binding modification comprises of the amino acidsequence of one of SEQ ID Nos: 1152-1157.

[0108] In an embodiment, the n-mer comprises of LHRLGPN (SEQ ID NO: 1392) and X1comprises of S, A, L, or M; X2 comprises of A or P; X3 comprises of Q; X4 comprises of A, E, F,H, I, L, M, N, P, Q, V, or Y; Xs comprises of Q; X6 comprises of A, P, S, or T; X7 comprises ofD, E, Q, or T; or any combination thereof. In an embodiment, the transferrin receptor bindingmodification comprises of the amino acid sequence of one of SEQ ID Nos: 1158-1210.

[0109] In an embodiment, the 7-mer comprises of FRSTNGV (SEQ ID NO: 1393),FVSTNGV (SEQ ID NO: 1394), FZ1STNGZ2 (SEQ ID NO: 1395), FRSTNGZ3 (SEQ ID NO:1396), or VESTNGR (SEQ ID NO: 1397) and wherein X1 comprises of S; X2 comprises of A, S,M, or D; X3 comprises of F, H, I, L, M, N, Q, R, Y, D, or E; X4 comprises of A, S, or M; Xscomprises of Q or P; X6 comprises of A, F, H, Q, or S; X, comprises of A, D, E, F, Q, S, or T; orany combination thereof. In an embodiment, wherein the transferrin receptor binding modificationcomprises of the amino acid sequence of one of SEQ ID Nos: 1211-1389.47

[0110] In an embodiment, the n-mer comprises of FRSTNGV (SEQ ID NO: 1393), and X1comprises of S; X2 comprises of A or S; X3 comprises of D; X4 comprises of A, or S; Xs comprisesof Q or P; X6 comprises of A, F, H, Q, or S; X, comprises of D, E, Q, or T; or any combinationthereof. In an embodiment, the transferrin receptor binding modification comprises of the aminoacid sequence of one of SEQ ID Nos: 1211-1266.

[0111] In an embodiment, the n-mer comprises of FVSTNGV (SEQ ID NO: 1394), and X1comprises of S; X2 comprises of A, S, or M; X3 comprises of Q, E, or D; X4 comprises of A, or M;Xs comprises of Q or P; X6 comprises of A; X7 comprises of E; or any combination thereof. In anembodiment, the transferrin receptor binding modification comprises of the amino acid sequenceof one of SEQ ID Nos: 1267-1298.

[0112] In an embodiment, the n-mer comprises of FZ1STNGZ2 (SEQ ID NO: 1395) orFRSTNGZ3 (SEQ ID NO: 1396), and X₁ comprises of S; X2 comprises of A or S; X3 comprises ofQ or D; X4 comprises of A; Xs comprises of Q; X6 comprises of A; X7 comprises of E; or anycombination thereof. In an embodiment, the transferrin receptor binding modification comprisesof the amino acid sequence of one of SEQ ID Nos: 1299-1319.

[0113] In an embodiment, the 7-mer comprises of VESTNGR (SEQ ID NO: 1397), and X1comprises of S; X2 comprises of S or D; X3 comprises of F, H, I, L, M, N, Q, R, or Y; X4 comprisesof A; Xs comprises of Q or P; X6 comprises of A; X7 comprises of A, D, E, F, Q, S, or T; or anycombination thereof. In an embodiment, the transferrin receptor binding modification comprisesof the amino acid sequence of one of SEQ ID Nos: 1320-1388.48WO 2025 / 155923Table C. EExample location of transferrin receptor (TfR1) binding modifications with example AAVsUPSTREAMSEOUEUENCEAAV1 VAVNFQ(SEQID NO: 1473)AAV2 VSTNLQ(SEQ ID ONO: 1474)AAV3 VANNLNLO(SEQ ID NO: 1475)AAV4 PGGDQ(SEQ ID NO: 1476)AAV5 MATNNО(SEQ ID NO: 1477)AAV6 VAVNLO(SEQ ID NO: 1478) (SEAAV7 VSSNLQ(SEQ ID NO: 1479)AAV8 VADNLQ(SEQ ID NO: 1480)AAV9 VATNHO(SEO ID NO: 1481)AAV10 VADNLQ(SEQ ID NO: 1482)AAV11 IADNNO(SEQ ID NO: 1483)AAV12 IADNNO(SEQ ID NO: 1484)AAV13 VSNNLQ(SEQ ID NO: 1485)7-mer INSERTION(X1-X2-X3-[7-mer]-X4-X5-X6-Х7)7-mer: YSRIGPN (SEQ ID NO: 1390), YSRNSDN (SEQ ID NO: 1391),LHRLGPN (SEQ ID NO:1392), FZISTNGZ2 (SEQ ID NO: 1393) 1392). FZ1STNGZ2 (SEQ ID NO: 1395), FVSTNGV (SEQ ID NO: 1394), FZ1STNGZ2(SEQ ID NO: 1395), FRSTNGZ3 (SEQ ID NO:1396), or VESTNGR (SEQ ID NO: 1397)X1, X2, X3, X4, X5, X6, X7 indicate one or moremodifications at amino acid positions in the capsidpolypeptide flanking the inserted 7-mer.The transferrin receptor binding modification caninclude any one of the amino acid sequences of SEQID Nos: 60-1210.DOWNSTREAMSEOUENCETGDVHAMG(SEQ ID NO: 1486)TADVNTQG(SEQ ID NO: 1487)TGTVNHỌG(SEO (SEQIDT ID NO: 1488)VDRLTALG(SEQ ID NO: 1489)TGTYNLOE(SEQ ID NO: 1490)TGDVHVMG(SEO ID NO: 1491)TQVVNNQG(SEQ ID NO: 1492)IGTVNSOG(SEO ID NO: 1493)TGWVONOG(SEO ID NO: 1494)VGNVNSQG(SEQ ID NO: 1495)TGNVTAMG(SEQID NO: 1496)IANLDAMG TAND(SEQ ID NO: 1497)TGTVNHỌG(SEO ID NO: 1498)49PCT / US2025 / 012207WO 2025 / 155923Table D. Example seququences includuing transnsferrin receptor (TfR1) binding modifications with example AAVsSEROTYPE STREAM 7-mer INSE ISERTION SEQUEENCEAAV1 VAVIIFOSSS YSRIGPN (SEQ (S ID(SEQ ID NO: 0: 1499) NO: 139 1390)AAV2 VSINLQRGN YSRIGPN (SEQ (S ID(SEQ ID NO: 0: 1500) 1 NO: 1390AAV3 VANNLOSS YSRIGPN (SEO (S ID(SECO ID NO: 1 0:501) NO: 1390)1AAV4 LYGGNSNS YSRIGPN (SEQ ID(SEQ ID NO: 1502) NO: 1390)AAV5 MATNNQSST YSRIGPN (SEQ ID(SEQ ID NO: 1503)AAV6 VAVNLMSSSNO: 13390)YSRIGPN (SEQ ID(SEQ ID NO: 1504) NO: 1390) 1AAV7 VSSNLOAAAAN(SEQ ID NO: 05) NO: 13390)AAV8 VADNLOOON(SEO ID NO: 1506)YSRIGPN (SEO IDNO: 1390)AAV9 VATNHOSASAO(SEO ID NO: O: 1507)AAV10 VADNLOOAN(SEQ ID NO: 1508)AAV11 IADNNQNATAAV12 IADNNONATAAV13VSNN(SEOID NO: 1511)(SEO ID NO: 1509)(SEOID NO: 1510)YSRIGPN (SEO IDYSRIGPN (SEO IDNO: 1390)3YSRIGPN (SEQ IDNO: 13390)YSRIGPN (SEQ IDNO: 1390)YSRIGPN (SEQ IDNO: 1390)YSRIGPN (SEQ IDNO: 1390)DOWNSTREAMSEQUENCEDPATGDVHA(SEQ ID NO: 1512)GQAATADVNT(SEQ ID NO: 1513)SAPTTGTVNH(SEO ID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515)TAPATGTYNLQE(SEQ ID NO: 1516)TDPATGDVHVMG(SEQ ID NO: 1517)TAAQTQVVNNOG(SEQ ID NO: 1518)TAPQIGTVNSQG(SEO ID NO: 1519)AQAQTGWVONOG(SEO ID NO: 1520)TGPIVGNVNSOG(SEQ ID NO: 1521)TAPITGNVTAMG(SEO ID NO: 1522)TAPHIANLDAMG(SEQ ID NO: 1523)AGPTTGTVNHỌG(SEQ ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEQ ID NO:)VAVIIFQSSSYSRIGPN(SEQ ID NO: 1525)VSINLQRGNYSRIGPN(SEQ ID NO: 1526)VANNLQSSNYSRIGPN(SEO ID NO: 1527)LYGGNSNSYSRIGPN(SEO ID NO: 1528)MATNNQSSTYSRIGPN(SEO ID NO: 1529)VAVNLMSSSYSRIGPN(SEO ID NO: 1530)VSSNLQAANYSRIGPN(SEQ ID NO: 1531)VADNLQQQNYSRIGPN(SEO ID NO: 1532)VATNHQSAQYSRIGPN(SEO ID NO: 1533)VADNLQQANYSRIGPN(SEQ ID NO: 1534)IADNNQNATYSRIGPN(SEO ID NO: 1535)IADNNONATYSRIGPN(SEQ ID NO: 1536)VSNNLONSNYSRIGPN(SEQ ID NO: 1537)7-mer INSERTION+DOWNSTREAM SEQUENCE(SEQ ID NO:)YSRIGPNDPATGDVHA(SEQ ID NO: 1538)YSRIGPNGQAATADVNT(SEQ ID NO: 1539)YSRIGPNSAPTTGTVNH(SEO ID NO: 1540)YSRIGPNNLPTVDRLTALG(SEQ ID NO: 1541)YSRIGPNTAPATGTYNLQE(SEQ ID NO: 1542)YSRIGPNTDPATGDVHVMG(SEQ ID NO: 1543)YSRIGPNTAAQTQVVNNQG(SEQ ID NO: 1544)YSRIGPNTAPQIGTVNSQG(SEO ID NO: 1545)YSRIGPNAQAQTGWVQNQG(SEO ID NO: 1546)YSRIGPNTGPIVGNVNSQG(SEQID NO: 1547)YSRIGPNTAPITGNVTAMG(SEO ID NO: 1548)YSRIGPNTAPHIANLDAMG(SEQ ID NO: 1549)YSRIGPNAGPTTGTVNHỌG(SEQID NO: 1550)50PCT / US2025 / 012207WO 2025 / 155923LYGGNSNS(SEQID NO: 1502)MAT(SEQ ID NO: 1503)VAVNLMSSSYSRNSDN (SEQID NO: 1391)YSRNSDN (SEQID NO: 1391)YSRNSDN (SEQGQAATADVNT(SEQ ID NO: 1513)SAPTTGTVNH SAPT(SEQ ID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515)TAPATGTYNLOE(SEO ID NO: 1516)TDPATGDVHVMG(SEQ ID NO: 1517)SEROTYPEPE UPSTREAM 7-mer INSERTION SEQUENCE DOWNSTREAM SEQUENCEAAVI VAVIIF IIFQSSS YSRNSDN (SEQ DPATGDVHA(SEQ IDID NO: 1499) ID NO: 1391) (SEQ ID NO: 1512)AAV2 VSINLOLORGN YSRNSDN (SEQ(SEO ID NO: 1500) ID NO: 1391)AAV3 VANNLOSSN YSRNSDN (SEQ(SEO ID NO: 1501 ID NO: 1391)AAV4AAV5AAV6AAV7 VSSNLQAANAAV8 VADNLQOQNAAV9 VATNHOSAOAAV10 VADNLOOANAAV11AAV12AAV13(SEQ ID NO: 1504)(SEQ ID NO: 1505)(SEO ID NO: 1506)(SEQ ID NO: 1507)(SEO ID NO: 1508)IADNNONAT(SEQ ID NO: 1509)IADNNONAT(SEQ ID NO: 1510)VSNNLQNSN(SEQ ID NO: 1511)ID NO: 1391)YSRNSDN (SEQID NO: 1391)YSRNSDN (SEQID NO: 1391)YSRNSDN (SEQID NO: 1391)YSRNSDN (SEQID NO: 1391)YSRNSDN (SEOID NO: 1391)YSRNSDN (SEQID NO: 1391)YSRNSDN (SEQID NO: 1391)TAAQTQVVNNQG(SEO ID NO: 1518)TAPOIGTVNSOG(SEO ID NO: 1519)AQAQTGWVONOG(SEQ ID NO: 1520)TGPIVGNVNSQG(SEQ ID NO: 1521)TAPITGNVTAMG(SEO ID NO: 1522)TAPHIANLDAMG(SEQ ID NO: 1523)AGPTTGTVNHQG(SEQ ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEQ ID NO:)VAVIIFQSSSYSRNSDN(SEQ ID NO: 1551)VSINLQRGNYSRNSDN(SEO ID NO: 1552)VANNLQSSNYSRNSDN(SEQ ID NO: 1553)LYGGNSNSYSRNSDN(SEQ ID NO: 1554)MATNNQSSTYSRNSDN(SEO ID NO: 1555)VAVNLMSSSYSRNSDN(SEO ID NO: 1556)VSSNLQAANYSRNSDN(SEO ID NO: 1557)VADNLQQQNYSRNSDN(SEO ID NO: 1558)VATNHỌSAQYSRNSDN(SEQ ID NO: 1559)VADNLQQANYSRNSDN(SEO ID NO: 1560)IADNNQNATYSRNSDN(SEO ID NO: 1561)IADNNQNATYSRNSDN(SEQ ID NO: 1562)VSNNLQNSNYSRNSDN(SEQ ID NO: 1563)7-mer INSERTION+DOWNSTREAM SEQUENCE STREAM(SEQ ID NO:)YSRNSDNDPATGDVHA(SEQ ID NO: 1564)YSRNSDNGQAATADVNT(SEO ID NO: 1565)YSRNSDNSAPTTGTVNH(SEQ ID NO: 1566)YSRNSDNNLPTVDRLTALG(SEQ ID NO: 1567)YSRNSDNTAPATGTYNLOE(SEO ID NO: 1568)YSRNSDNTDPATGDVHVMG(SEQ ID NO: 1569)YSRNSDNTAAQTQVVNNQG(SEQ ID NO: 1570)YSRNSDNTAPQIGTVNSQG(SEO ID NO: 1571)YSRNSDNAQAQTGWVQNQG(SEQ ID NO: 1572)YSRNSDNTGPIVGNVNSQG(SEO ID NO: 1573)YSRNSDNTAPITGNVTAMG(SEO ID NO: 1574)YSRNSDNTAPHIANLDAMG(SEQ ID NO: 1575)YSRNSDNAGPTTGTVNHQG(SEQ ID NO: 1576)51PCT / US2025 / 012207WO 2025 / 155923(SEQ ID NO: 1503)DOWNSTREAMGQAATADVNT(SEQ ID NO: 1513)SAPTTGTVNH SAPTTO (SEQ ID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515)TAPATGTYNLOE(SEO ID NO: 1516)TDPATGDVHVMG(SEQ ID NO: 1517)UPSTREAM 7-mer INSERTION SEROTYPE SEQUENCE SEQUENCEAAVI VAVIIFIIFOSSS LHRLGPN (SEQ DPATGDVHA(SEQ ID NO: 1499) ID NO: 1392) (SEQ ID NO: 1512)AAV2 VSINLNLQRGN LHRLGPN (SEQ(SEO ID NO: 1500) ID NO: 1392)AAV3 VANNLOSSOSN LHRLGPN (SEQ(SEQ ID NO: 1501) 1 ID NO: 1392)AAV4 LYGGNSNS LHRLGPN (SEQ(SEQO ID NO: 1502) ID NO: 1392)AAV5 MATNNOSST LHRLGPN (SEQID NO: 1392)AAV6 VAVNLMSSS LHRLGPN (SEQAAV7 VSSNLQAAN LHRLGPN (SEQID NO: 1392)AAV8 VADNLOOON LHRLGPN (SEQID NO: 1392)AAV9 VATNHQSAQ LHRLGPN (SEQID NO: 1392)AAVAV10AAVI1AAV12AV13(SEQID NO: 1504)(SEQ ID NO: 1505)(SEO ID NO: 1506)(SEQID NO: 1507)VADNLOOAN(SEO ID NO: 1508)IADNNONAT(SEQ ID NO: 1509)IADNNONAT(SEQID NO: 1510)VSNNLQNSN(SEQ ID NO: 1511)ID NO: 1392)LHRLGPN (SEQID NO: 1392)LHRLGPN (SEOID NO: 1392)LHRLGPN (SEQID NO: 1392)LHRLGPN (SEQID NO: 1392)TAAQTQVVNNQG(SEQ ID NO: 1518)TAPQIGTVNSOG(SEO ID NO: 1519)AQAQTGWVONOG(SEQ ID NO: 1520)TGPIVGNVNSQG(SEQ ID NO: 1521)TAPITGNVTAMG(SEQ ID NO: 1522)TAPHIANLDAMG(SEQ ID NO: 1523)AGPTTGTVNHQG(SEO ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEO ID NO:)VAVIIFQSSSLHRLGPN(SEQ ID NO: 1577)VSINLORGNLHRLGPN(SEO ID NO: 1578)VANNLQSSNLHRLGPN(SEQ ID NO: 1579)LYGGNSNSLHRLGPN(SEQ ID NO: 1580)MATNNQSSTLHRLGPN(SEO ID NO: 1581)VAVNLMSSSLHRLGPN(SEO ID NO: 1582)VSSNLQAANLHRLGPN(SEQ ID NO: 1583)VADNLQQQNLHRLGPN(SEO ID NO: 1584)VATNHQSAQLHRLGPN(SEQ ID NO: 1585)VADNLQQANLHRLGPN(SEO ID NO: 1586)IADNNQNATLHRLGPN(SEO ID NO: 1587)IADNNQNATLHRLGPN(SEQ ID NO: 1588)VSNNLQNSNLHRLGPN(SEQ ID NO: 1589)7-mer INSERTION+DOWNSTREAM SEQUENCE EAM(SEQ ID NO:)LHRLGPNDPATGDVHA(SEQ ID NO: 1590)LHRLGPNGQAATADVNT(SEO ID NO: 1591)LHRLGPNSAPTTGTVNH(SEQ ID NO: 1592)LHRLGPNNLPTVDRLTALG(SEQ ID NO: 1593)LHRLGPNTAPATGTYNLQE(SEO ID NO: 1594)LHRLGPNTDPATGDVHVMG(SEQ ID NO: 1595)LHRLGPNTAAQTQVVNNQG(SEQ ID NO: 1596)LHRLGPNTAPQIGTVNSQG(SEQ ID NO: 1597)LHRLGPNAQAQTGWVQNQG(SEQ ID NO: 1598)LHRLGPNTGPIVGNVNSQG(SEQ ID NO: 1599)LHRLGPNTAPITGNVTAMG(SEO ID NO: 1600)LHRLGPNTAPHIANLDAMG(SEQ ID NO: 1601)LHRLGPNAGPTTGTVNHQG(SEQ ID NO: 1602)52PCT / US2025 / 012207WO 2025 / 155923 SEQUENCE FRSTNGV (SEO ID NO: 1393) FRSTNGV (SEQ ID NO: 1393)UPSTREAM 7-mer INSERTION SEROTYPEPEAAVI VAVIIFO(SEQ IDNONO: 1499)AAV2 VSINLOR(SEO ID NO: 0: 1500)AAV3 VANNLOSSNAAV4(SEQ ID NO: 1501)LYGGNSNS(SEQ ID NO: 1502)AAV5 MAIATNNOSST(SEQ ID NO: 1503)AAV6 VAVNLMSSSAAV (SEQ ID NO: 1504) VSSNLQAAN(SEQ ID NO: 1505)VADNLQOQN(SEO ID NO: 1506)VATNHQSAQ(SEQ ID NO: 1507)VADNLQQAN(SEO ID NO: 1508)AAV8AAV9AAV10AAVI1AAV12AAV13IADNNONAT(SEQVSNNLQNSNID NO: 1509)IADNNQNAT(SEQ ID NO: 1510)(SEQ IDNO: 1511)FRSTNGV (SEQID NO: 1393)FRSTNGV (SEQID NO: 1393)FRSTNGV (SEOID NO: 1393)FRSTNGV(SEQID NO: 1393)FRSTNGV (SEQID NO: 1393)FRSTNGV (SEQID NO: 1393)FRSTNGV (SEQID NO: 1393)FRSTNGV(SEQID NO: 1393)ID NO: 1393)DOWNSTREAMSEQUENCEDPATGDVHA(SEQ ID NO: 1512)GQAATADVNT(SEQ ID NO: 1513)SAPTTGTVNH SAPT(SEQ ID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515)TAPATGTYNLOE(SEO ID NO: 1516)TDPATGDVHVMG(SEQ ID NO: 1517)TAAQTQVVNNQG(SEQ ID NO: 1518)TAPQIGTVNSOG(SEO ID NO: 1519)AQAQTGWVONOG(SEQ ID NO: 1520)TGPIVGNVNSQG(SEQ ID NO: 1521)TAPITGNVTAMG(SEO ID NO: 1522)FRSTNGV (SEQFRSTNGV (SEQID NO: 1393)FRSTNGV (SEQTAPHIANLDAMG(SEQ ID NO: 1523)AGPTTGTVNHQGID NO: 1393) (SEO ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEQ ID NO:)VAVIIFQSSSFRSTNGV(SEQ ID NO: 1603)VSINLQRGNFRSTNGV(SEO ID NO: 1604)VANNLQSSNFRSTNGV(SEQID NO: 1605)LYGGNSNSFRSTNGV(SEQ ID NO: 1606)MATNNQSSTFRSTNGV(SEO ID NO: 1607)VAVNLMSSSFRSTNGV(SEO ID NO: 1608)VSSNLQAANFRSTNGV(SEQ ID NO: 1609)VADNLQQQNFRSTNGV(SEO ID NO: 1610)VATNHQSAQFRSTNGV(SEQ ID NO: 1611)VADNLQQANFRSTNGV(SEO ID NO: 1612)IADNNQNATFRSTNGV(SEQ ID NO: 1613)IADNNQNATFRSTNGV(SEQ ID NO: 1614)VSNNLQNSNFRSTNGV(SEQ ID NO: 1615)7-mer INSERTION+DOWNSTREAM SEQUENCE(SEQ ID NO:)FRSTNGVDPATGDVHA(SEQ ID NO: 1616)FRSTNGVGQAATADVNT(SEO ID NO: 1617)FRSTNGVSAPTTGTVNH(SEQ ID NO: 1618)FRSTNGVNLPTVDRLTALG(SEQ ID NO: 1619)FRSTNGVTAPATGTYNLQE(SEO ID NO: 1620)FRSTNGVTDPATGDVHVMG(SEQ ID NO: 1621)FRSTNGVTAAQTQVVNNQG(SEQ ID NO: 1622)FRSTNGVTAPQIGTVNSQG(SEO ID NO: 1623)FRSTNGVAQAQTGWVQNQG(SEQ ID NO: 1624)FRSTNGVTGPIVGNVNSQG(SEO ID NO: 1625)FRSTNGVTAPITGNVTAMG(SEQ ID NO: 1626)FRSTNGVTAPHIANLDAMG(SEQ ID NO: 1627)FRSTNGVAGPTTGTVNHQG(SEQ ID NO: 1628)53PCT / US2025 / 012207WO 2025 / 155923(SEQ ID NO: 1501) LYGGNSNS(SEQ ID NO: 15002)МАTNNOSS(SEO ID NO: 1503)VAVNLMSSSFVSTNGV(SEQID NO: 1394)FVSTNGV (SEQID NO: 1394)FVSTNGV (SEOID NO: 1394)FVSTNGV (SEQDOWNSTREAMSEOUENCEGQAATADVNT(SEO ID NO: 1513)SAPTTGTVNH SA(SEQ ID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515)TAPATGTYNLOE(SEO ID NO: 1516)TDPATGDVHVMGUPSTREAM 7-mer INSERTION SEROTYPE SEQUENCEAAVI VAVIIFOFSSS FVSTNGV (SEQ DPATGDVHA(SEQ ID NO: 1499) ID NO: 1394) (SEQ ID NO: 1512)AAV2 VSINLORGNGN FVSTNGV (SEQ(SEO ID NO: 1500 600) ID NO: 1394)AAV3 VANNLOSSNAAV4AAV5AAV6AAV7AAV8AAV9AAV10AAVI1AAV12AAV13(SEQVSNNLQNSN ID NO: 1504)VSSNLQAAN(SEQ ID NO: 1505)VADNLQOON(SEQ ID NO: 1506)VATNHQSAQ(SEQ ID NO: 1507)VADNLQQAN(SEO ID NO: 1508)IADNNONAT(SEQ ID NO: 1509)IADNNONAT(SEQ ID NO: 1510)(SEQ ID NO: 1511)ID NO: 1394)FVSTNGV (SEQID NO: 1394)FVSTNGV(SEQID NO: 1394)FVSTNGV(SEQID NO: 1394)FVSTNGV(SEQID NO: 1394)FVSTNGV (SEOID NO: 1394)FVSTNGV (SEQID NO: 1394)FVSTNGV(SEQ(SEQ ID NO: 1517)TAAQTQVVNNQG(SEQ ID NO: 1518)TAPQIGTVNSOG(SEO ID NO: 1519)AQAQTGWVONOG(SEQID NO: 1520)TGPIVGNVNSQG(SEQ ID NO: 1521)TAPITGNVTAMG(SEQ ID NO: 1522)TAPHIANLDAMG(SEQ ID NO: 1523)AGPTTGTVNHQGID NO: 1394) (SEQ ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEO ID NO:)VAVIIFQSSSFVSTNGV(SEQ ID NO: 1629)VSINLQRGNFVSTNGV(SEO ID NO: 1630)VANNLQSSNFVSTNGV(SEQID NO: 1631)LYGGNSNSFVSTNGV(SEQ ID NO: 1632)MATNNQSSTFVSTNGV(SEO ID NO: 1633)VAVNLMSSSFVSTNGV(SEO ID NO: 1634)VSSNLQAANFVSTNGV(SEO ID NO: 1635)VADNLQQQNFVSTNGV(SEO ID NO: 1636)VATNHQSAQFVSTNGV(SEQ ID NO: 1637)VADNLQQANFVSTNGV(SEO ID NO: 1638)IADNNQNATFVSTNGV(SEO ID NO: 1639)IADNNQNATFVSTNGV(SEQ ID NO: 1640)VSNNLQNSNFVSTNGV(SEQID NO: 1641)7-mer INSERTION+DOWNSTREAM SEQUENCE SED (SEQ ID NO:)FVSTNGVDPATGDVHA(SEQ ID NO: 1642)FVSTNGVGQAATADVNT(SEO ID NO: 1643)FVSTNGVSAPTTGTVNH(SEQ ID NO: 1644)FVSTNGVNLPTVDRLTALG(SEQ ID NO: 1645)FVSTNGVTAPATGTYNLQE(SEO ID NO: 1646)FVSTNGVTDPATGDVHVMG(SEO ID NO: 1647)FVSTNGVTAAQTQVVNNQG(SEO ID NO: 1648)FVSTNGVTAPQIGTVNSQG(SEQ ID NO: 1649)FVSTNGVAQAQTGWVONOG(SEQ ID NO: 1650)FVSTNGVTGPIVGNVNSQG(SEO ID NO: 1651)FVSTNGVTAPITGNVTAMG(SEO ID NO: 1652)FVSTNGVTAPHIANLDAMG(SEQ ID NO: 1653)FVSTNGVAGPTTGTVNHQG(SEQ ID NO: 1654)54PCT / US2025 / 012207WO 2025 / 155923MATNNOSST(SEQ ID NO: 1503)FZ1STNGZ2 (SEQID NO: 1395)FZ1STNGZ2 (SEQDOWNSTREAMSEOUENCEGQAATADVNT(SEO ID NO: 1513)SAPTTGTVNH SA(SEO ID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515)TAPATGTYNLOE(SEO ID NO: 1516)TDPATGDVHVMG(SEQ ID NO: 1517)UPSTREAM 7-mer INSERTION SEROTYPE SEQUENCEAAVI VAVIIFIIFQSSS(SEQ IDD NO: 1499)FZ1STNGZ2 (SEQID NO: 1395)DPATGDVHA(SEQ ID NO: 1512)AAV2 VSININLORGN FZ1STNGZ2 (SEQSEQ ID NO: 1500) ID NO: 1395)AAV3 VANNLOSSN FZ1STNGZ2 (SEQ(SEQ ID NO: 1501) ID DNNO: 1395)AAV4 LYGGNSNS FZ1STNGZ2 (SEQ(SEQ ID NO: 1502) ID NO: 1395)AAV5AAV6 VAVNLMSSSAAV7 VSSNLQAANAAV8 VADNLQOONAAV9AAV10AAV11AAV12AAV13(SEQ ID NO: 1504)(SEQ ID NO: 1505)(SEO ID NO: 1506)VATNHQSAQ(SEQ ID NO: 1507)VADNLQQAN(SEO ID NO: 1508)IADNNONAT(SEQ ID NO: 1509)IADNNONAT(SEQ ID NO: 1510)VSNNLQNSN(SEQ ID NO: 1511)ID NO: 1395)FZ1STNGZ2 (SEQID NO: 1395)FZISTNGZ2 (SEQID NO: 1395)FZ1STNGZ2 (SEQID NO: 1395)FZ1STNGZ2 (SEQID NO: 1395)FZ1STNGZ2 (SEQID NO: 1395)FZ1STNGZ2 (SEQID NO: 1395)FZ1STNGZ2 (SEQID NO: 1395)TAAQTQVVNNQG(SEQ ID NO: 1518)TAPQIGTVNSOG(SEOID NO: 1519)AQAQTGWVONOG(SEQ ID NO: 1520)TGPIVGNVNSQG(SEQ ID NO: 1521)TAPITGNVTAMG(SEO ID NO: 1522)TAPHIANLDAMG(SEQ ID NO: 1523)AGPTTGTVNHQG(SEQ ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEQ ID NO:)VAVIIFQSSSFZ1STNGZ2(SEQ ID NO: 1655)VSINLQRGNFZ1STNGZ2(SEO ID NO: 1656)VANNLQSSNFZ1STNGZ2(SEQ ID NO: 1657)LYGGNSNSFZ1STNGZ2(SEQ ID NO: 1658)MATNNQSSTFZISTNGZ2(SEO ID NO: 1659)VAVNLMSSSFZ1STNGZ2(SEO ID NO: 1660)VSSNLQAANFZ1STNGZ2(SEQ ID NO: 1661)VADNLQQQNFZ1STNGZ2(SEO ID NO: 1662)VATNHOSAOFZ1STNGZ2(SEQ ID NO: 1663)VADNLQQANFZISTNGZ2(SEQ ID NO: 1664)IADNNONATFZ1STNGZ2(SEO ID NO: 1665)IADNNONATFZISTNGZ2(SEQ ID NO: 1666)VSNNLQNSNFZ1STNGZ2(SEO ID NO: 1667)7-mer INSERTION+DOWNSTREAM SEQUENCE TREAM SE(SEQ ID NO:)FZ1STNGZ2DPATGDVHА(SEQ ID NO: 1668FZ1STNGZ2GQAATADVNT(SEO ID NO: 1669)FZ1STNGZ2SAPTTGTVNH(SEQ ID NO: 1670)FZ1STNGZ2NLPTVDRLTALG(SEQ ID NO: 1671)FZ1STNGZ2TAPATGTYNLQE(SEO ID NO: 1672)FZ1STNGZ2TDPATGDVHVMG(SEQ ID NO: 1673)FZ1STNGZ2TAAQTQVVNNQG(SEQ ID NO: 1674)FZ1STNGZ2TAPQIGTVNSQG(SEO ID NO: 1675)FZ1STNGZ2AQAQTGWVQNQG(SEQ ID NO: 1676)FZ1STNGZ2TGPIVGNVNSQG(SEO ID NO: 1677)FZ1STNGZ2TAPITGNVTAMG(SEO ID NO: 1678)FZ1STNGZ2TAPHIANLDAMG(SEQ ID NO: 1679)FZ1STNGZ2AGPTTGTVNHQG(SEQ ID NO: 1680)Z1 is selected from the group consisting of A, D, H, N, Q, and S; Z2 is selected from the group consisting of K and R.55PCT / US2025 / 012207WO 2025 / 155923 FRSTNGZ3 (SEQ ID NO: 1396) FRSTNGZ3 (SEQ ID NO:0: 1396) FRSTNGZ3 (SEQ ID NO: 1396) UPSTREAM 7-mer INSERTION ROTYPE SEQUENCE AAV1 VAVII VIIFOSSS (SEO ID ID NO: 1499) AAV2 VSINLQROORGN (SEO ID NO 1500) AAV3 VANNLOSOSSN (SEO ID NO: 1501)AAV4 LYGGNGNSNS FRSTNGZ3 (SEQ(SEQ II ID NO: 1502) ID NO: 1396)AAV5 MATNNQSST FRSTNGZ3 (SEQ(SEQ ID NO: 1503) ID NO: 1396)AAV6 VAVNLMSSS FRSTNGZ3 (SEQID NO: 1396)AAV7 VSSNLOAAN FRSTNGZ3 (SEQID NO: 1396)AAV8 VADNLQQON FRSTNGZ3 (SEQID NO: 1396)AAV9 VATNHOSAO FRSTNGZ3 (SEOAAV10AAV11AAVI2AAV13(SEQ ID NO: 1504)(SEO ID NO: 1505)(SEQ ID NO: 1506)(SEO ID NO: 1507)VADNLOOAN(SEO ID NO: 1508)IADNNQNAT(SEQ ID NO: 1509)IADNNONAT(SEQ ID NO: 1510)VSNNLONSN(SEO ID NO: 1511)ID NO: 1396)FRSTNGZ3 (SEQID NO: 1396)FRSTNGZ3 (SEQID NO: 1396)FRSTNGZ3 (SEQID NO: 1396)FRSTNGZ3 (SEQID NO: 1396)Z3 is selected from the group consisting of L, M, and R.DOWNSTREAMSEQUENCEDPATGD VHA(SEO ID NO: 1512)GQAATADVNT(SEQ ID NO: 1513)SAPTTGTVNH(SEQ ID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515) (SEQID NOTAPATGTYNLQE(SEQ ID NO: 1516)TDPATGDVHVMG(SEQ ID NO: 1517)TAAQTQVVNNQG AAQ(SEO ID NO: 1518)TAPQIGTVNSQG(SEQ ID NO: 1519)AQAQTGWVQNOG(SEQ ID NO: 1520)TGPIVGNVNSOG(SEQ ID NO: 1521)TAPITGNVTAMG(SEQ ID NO: 1522)TAPHIANLDAMG(SEQ ID NO: 1523)AGPTTGTVNHQG(SEQ ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEO ID NO:)VAVIIFOSSSFRSTNGZ3(SEO ID NO: 1681)VSINLQRGNFRSTNGZ3(SEQ ID NO: 1682) VANNLQSSNFRSTNGZ3(SEQ ID NO: 1683)LYGGNSNSFRSTNGZ3(SEQ ID NO: 1684)MATNNQSSTFRSTNGZ3(SEQ ID NO: 1685)VAVNLMSSSFRSTNGZ3(SEQ ID NO: 1686)VSSNLQAANFRSTNGZ3(SEO ID NO: 1687)VADNLQQQNFRSTNGZ3(SEQ ID NO: 1688)VATNHQSAQFRSTNGZ3(SEO ID NO: 1689)VADNLQQANFRSTNGZ3(SEQ ID NO: 1690)IADNNQNATFRSTNGZ3(SEQ ID NO: 1691)IADNNONATFRSTNGZ3(SEO ID NO: 1692)VSNNLONSNFRSTNGZ3(SEO ID NO: 1693)7-mer INSERTION+DOWNSTREAM SEQUENCE(SEQ ID NO:)FRSTNGZ3DPATGDVHA(SEO ID NO: 1694)FRSTNGZ3GQAATADVNT(SEQ ID NO: 1695)FRSTNGZ3SAPTTGTVNH(SEO ID NO: 1696)FRSTNGZ3NLPTVDRLTALG(SEO ID NO: 1697)FRSTNGZ3TAPATGTYNLQE(SEQ ID NO: 1698)FRSTNGZ3TDPATGDVHVMG(SEQ ID NO: 1699)FRSTNGZ3TAAQTQVVNNQG(SEO ID NO: 1700)FRSTNGZ3TAPQIGTVNSQG(SEQ ID NO: 1701)FRSTNGZ3AQAQTGWVQNQG(SEO ID NO: 1702)FRSTNGZ3TGPIVGNVNSOG(SEO ID NO: 1703)FRSTNGZ3TAPITGNVTAMG(SEQ ID NO: 1704)FRSTNGZ3TAPHIANLDAMG(SEO ID NO: 1705)FRSTNGZ3AGPTTGTVNHOG(SEQ ID NO: 1706)56PCT / US2025 / 012207WO 2025 / 155923 ID NO: 1397) VESTNGR (SEQ ID NO: 1397) VESTNGR (SEQID NO: 1397)VESTNGR (SEQDOWNSTREAMSEOUENCE(SEQ ID NO: 1512)GQAATADVNT(SEO ID NO: 1513)SAPTTGTVNH SAPT(SEQID NO: 1514)NLPTVDRLTALG(SEQ ID NO: 1515)TAPATGTYNLOEUPSTREAM 7-mer INSERTION SEROTYPE SEQUENCEAAVI OSSS VESTNGR (SEQ DPATGDVHA(SEQ ID NO: 1499) ID NO: 1397)AAV2 VSINLQRORGN VESTNGR (SEQ(SEO ID NO:O: 1500)AAV3 VANNLOSSN(SEQ ID NO: 150 1501)AAV4 LYGGNSNS(SEQ ID NO: 1502)AAV5 MATATNNOSST(SEO ID NO: 1503) ID NO: 1397)AAV6 VAVNLMSSS VESTNGR (SEQID NO: 1397)V7 VSSNLQAAN VESTNGR (SEQID NO: 1397)AAV8 VADNLQOON VESTNGR (SEQID NO: 1397)AAV9 VATNHQSAQ VESTNGR (SEQID NO: 1397)AAV10 VESTNGR (SEQID NO: 1397)AAV11 VESTNGR (SEQAAV12AAV13(SEQ ID NO: 1504)(SEQ ID NO: 1505)(SEO ID NO: 1506)(SEQ ID NO: 1507)VADNLQQAN(SEO ID NO: 1508)IADNNONAT(SEQ ID NO: 1509)IADNNONAT(SEQ ID NO: 1510)VSNNLQNSN(SEQ ID NO: 1511)ID NO: 1397)VESTNGR(SEQID NO: 1397)VESTNGR (SEQID NO: 1397)(SEO ID NO: 1516)TDPATGDVHVMG(SEQ ID NO: 1517)TAAQTQVVNNQG(SEQ ID NO: 1518)TAPQIGTVNSOG(SEO ID NO: 1519)AQAQTGWVQNQG(SEQ ID NO: 1520)TGPIVGNVNSQG(SEO ID NO: 1521)TAPITGNVTAMG(SEO ID NO: 1522)TAPHIANLDAMG ТАРҢ(SEQ ID NO: 1523)AGPTTGTVNHQG(SEQ ID NO: 1524)UPSTREAM SEQUENCE +7-mer INSERTION(SEQ ID NO:)VAVIIFQSSSVESTNGR(SEQ ID NO: 1707)VSINLQRGNVESTNGR(SEO ID NO: 1708)VANNLQSSNVESTNGR(SEQ ID NO: 1709)LYGGNSNSVESTNGR(SEQ ID NO: 1710MATNNQSSTVESTNGR(SEO ID NO: 1711)VAVNLMSSSVESTNGR(SEO ID NO: 1712)VSSNLQAANVESTNGR(SEQ ID NO: 1713)VADNLQQQNVESTNGR(SEO ID NO: 1714)VATNHOSAOVESTNGR(SEQ ID NO: 1715)VADNLQQANVESTNGR(SEO ID NO: 1716)IADNNQNATVESTNGR(SEO ID NO: 1717)|IADNNQNATVESTNGR(SEQ ID NO: 1718)VSNNLQNSNVESTNGR(SEQ ID NO: 1719)7-mer INSERTION+DOWNSTREAM SEQUENCE REAM SE(SEQ ID NO:)VESTNGRDPATGDVHA(SEQ ID NO: 1720)|VESTNGRGQAATADVNT(SEO ID NO: 1721)VESTNGRSAPTTGTVNH VE(SEQ ID NO: 1722)VESTNGRNLPTVDRLTALG(SEQ ID NO: 1723)VESTNGRTAPATGTYNLQE(SEO ID NO: 1724)VESTNGRTDPATGDVHVMG(SEQ ID NO: 1725)VESTNGRTAAQTQVVNNQG(SEQ ID NO: 1726)VESTNGRTAPQIGTVNSQG(SEQ ID NO: 1727)VESTNGRAQAQTGWVQNQG(SEQ ID NO: 1728)VESTNGRTGPIVGNVNSQG(SEO ID NO: 1729)VESTNGRTAPITGNVTAMG(SEQ ID NO: 1730)VESTNGRTAPHIANLDAMG(SEQ ID NO: 1731)VESTNGRAGPTTGTVNHQG(SEQ ID NO: 1732)57PCT / US2025 / 012207

[0114] In an embodiment, in addition to the n-mer motif(s), the transferrin receptor bindingmodification can include a polypeptide, a polynucleotide, a lipid, a polymer, a sugar, orcombination thereof.a

[0115] The engineered viral capsid and / or capsid proteins can be encoded by one or moreengineered viral capsid polynucleotides. In an embodiment, the engineered viral capsidpolynucleotide is an engineered AAV capsid polynucleotide or engineered adenovirus capsidpolynucleotide. In an embodiment, an engineered viral capsid polynucleotide (e.g., an engineeredAAV capsid polynucleotide or engineered adenovirus capsid polynucleotide) can include a 3'polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

[0116] In an embodiment, the engineered polynucleotide can be included in a polynucleotidethat is configured to express the engineered capsid in a host cell system for production of viralparticles. The host cell system may also include a construct that expresses a recombinant viralgenome that comprises a transgene encoding a polypeptide or nucleic acid operably linked to oneor more regulatory sequences that promote expression of the transgene in a target cell, including arecombinant AAV genome where the transgene and regulatory sequences are flanked by AAVITR sequences.

[0117] In an embodiment, the engineered AAV capsid encoding polynucleotide can beincluded in a polynucleotide that is configured to express the engineered capsid in a host cellsystem for production of AAV viral particles. The host cell system may also include a constructthat expresses a recombinant AAV viral genome that comprises a transgene encoding apolypeptide or nucleic acid operably linked to one or more regulatory sequences that promoteexpression of the transgene in a target cell, including a recombinant AAV genome where thetransgene and regulatory sequences are flanked by AAV ITR sequences. In an embodiment, theengineered AAV capsid encoding polynucleotide can be operably coupled to a polyadenylationtail. In an embodiment, the polyadenylation tail can be an SV40 polyadenylation tail. In anembodiment, the AAV capsid encoding polynucleotide can be operably coupled to a promoter. Inan embodiment, the regulatory sequence that regulates the expression of the transgene is apromoter and can be a tissue- or cell type-specific promoter. In an embodiment, the tissue-specificpromoter is specific for muscle (e.g., cardiac, skeletal, and / or smooth muscle), neurons or othernervous system cells (e.g., astrocytes, glial cells, Schwann cells, ependymal cells, pericyte,oligodendrocyte, oligodendrocyte progenitor), specific neuronal subtype (e.g, dopaminergic58neuron; Purkinje Cell; Parvalbumin, somatostatin, VIP inhibitory neuron; medium spiny neuron,Pyramidal neuron, motor neuron, etc.), endothelial cell, fat, spleen, liver, kidney, immune cells,synovial fluid cells, skin cells, cartilage, tendons, connective tissue, bone, pancreas, adrenal gland,blood cell, bone marrow cells, placenta, endothelial cells, and combinations thereof. In anembodiment, the promoter can be a constitutive promoter. Suitable tissue specific promoters andconstitutive promoters are discussed elsewhere herein and are generally known in the art and canbe commercially available. Suitable neuronal tissue / cell specific promoters include, but are notlimited to, GFAP promoter (astrocytes), SYN1 promoter (neurons), and NSE / RU5' (matureneurons). In an embodiment, the regulatory sequence that regulates the expression of the transgeneis a promoter and can be a cell state regulating promotor or drug inducible promotor.

[0118] 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 anoperatively-linked transgene in neurons as compared to expression in non-neuronal cells. Nonlimiting example of tissue-specific expression elements for neurons include neuron-specificenolase (NSE) (see, e.g., EMBL HSEN02, X51956); an aromatic amino acid decarboxylase(AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsinpromoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al,(1987) Cell, 51 :7-19; Llewellyn et al. (2010) Nat. Med., 16(10):1161-1 166); a serotonin receptorpromoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al.,(2009) Gene Ther., 16:437; Sasaoka et al., (1992) Mol. Brain Res., 16:274; Boundy et al., (1998)J. Neurosci., 18:9989; and Kaneda et al., (1991) Neuron, 6:583-594); a methyl-CpG bindingprotein 2 (MeCP2) promoter, an optimized methyl- CpG binding protein 2 (MeCP2) promoter (thepublished International Patent Application No. WO2020180928, the content of which isincorporated by reference herein in its entirety), a Ca2+-calmodulin-dependent protein kinase IIalpha (CaMKIIα) promoter (see, e.g., Mayford et al., (1996) Proc. Natl. Acad. Sci. USA, 93:13250;and Casanova et al., (2001) Genesis, 31 :37); a GnRH promoter (see, e.g., Radovick et al., (1991)Proc. Natl. Acad. Sci. USA, 88:3402- 3406); an L7 promoter (see, e.g., Oberdick et al., (1990)Science, 248:223-226); a DNMT promoter (see, e.g., Badge et al., (1988) Proc. Natl. Acad. Sci.USA, 85:3648-3652); an enkephalin promoter (see, e.g., Comb et al., (1988) EMBO J., 17:3793-3805); a myelin basic protein (MBP) promoter; a CMV enhancer / platelet-derived growth factor-p59promoter (see, e.g., Liu et al., (2004) Gene Ther., 11 :52-60); and the like. In some aspects, aportion of or all the minimal human synapsin 1 promoter (SYN) can be used (Kugler et al., (2003)Gene Ther., 10(4): 337-47; Thiel et al, (1991) Proc. Natl. Acad. Sci. USA, 88(8) 3431 -5; Castleet al., (2016) Methods Mol. Biol., 1382: 133-49; McLean et al., (2014) Neurosci. Lett., 576: 73-78; Kugler et al., (2003) Virology, 311 (1): 89-95). In other aspects, the neural-specific promotercan be mGluR2, NFL, NFH, nẞ2, PPE, Enk and EAAT2 promoters. A non-limiting example of atissue-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 foroligodendrocytes include the myelin basic protein (MBP) promoter. In certain aspects, a neuronalpromoter 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 isincorporated by reference herein in its entirety). In one aspect, the promoter can be a fugu SST(somatostatin) promoter (Nathanson, et al. Frontiers in Neural Circuits 3: 19). Examples of retinalspecific promoters include, but are not limited to, NA65p (RPE cells), Nefh (ganglion cells),hGRK1 (rod and cone photoreceptor cells), hRLBP1 (Müller glial cells and RPE cells), humanRHO (rhodopsin), human rhodopsin kinase (RHOK / GRK1) (an exemplary list of retina cellspecific promoters can be found in Buck et al. (2020) International Journal of Molecular Sciences21 (12), the content of which is incorporated by reference in its entirety). Non-limiting examplesof liver promoters include hAAT and TBG. Non-limiting examples of skeletal muscle promotersinclude Desmin, MCK and C5-12. Additional exemplary tissue-specific promoters can be foundin the TiProD (Tissue specific promoter database webpage tiprod.bioinf.med.uni-goettingen.de).

[0119] In other aspects, a promoter can be an inducible promoter (i.e., a promoter whoseactivity is controlled by an external stimulus, e.g., the presence of a particular temperature,compound, or protein). In some aspects, a promoter may be a temporally restricted promoter thatdrives 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 biologicalprocess. Prokaryotic (Gossen et al. TIBS 18: 471475, 1993) and insect regulatory systems (No etal. Proc. Natl. Aced. Sci. USA 93: 3346-3351, 1996) have been adapted to construct gene switchesthat function in mammalian cells. Since inducer molecules are not expected to have targets inmammalian cells, the possibility of interference with cellular processes is reduced. Of theprokaryotic proteins, the repressors from the lac operon (Brown, M., et at. Cell 49: 603-612, 1987;60and Hu, M. C. -T. and N. Davidson Cell 48: 555-566, 1987), the tet operon (e.g., U.S. Patent No.7,541,446, the content of which is incorporated by reference herein in its entirety) and the cumateoperon (e.g., U.S. Patent No. 7,745,592, the content of which is incorporated by reference hereinin its entirety) have been shown to function in mammalian cells. Many have been incorporated ineukaryotic inducible expression systems using different strategies to control activation andrepression of expression. Activation of expression is mediated by a chimeric transactivator proteinformed by the fusion of the bacterial repressor with an activation domain (Gossen, M. and H.Bujard, Proc. Natl. acad. sci. USA 89: 5547-5551, 1992, and Gossen, M., et al. Science 268: 1766-1769, 1995; U.S. Patent No.7,745,592, the contents of which are incorporated by reference hereinin their entireties). The transactivator can activate transcription when bound to its DNA recognitionsequence placed upstream of the minimal promoter. The ability of the activator to bind DNA isdependent on the presence / absence of the inducer molecule (e.g., doxycycline or cumatedepending on the inducible system being used). Repression of expression is mediated by therepressor bound to operator sites placed downstream of the minimal promoter in the absence ofinducer and repression is relieved on the addition of the inducer (Brown, M., et al. Cell 49: 603-612, 1987). In one aspect, the promoter may bea promoter which is less than 1 kb. The promotermay 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 between200-300, 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 or700-800 nucleotides. In one aspect, the promoter can be a pol III-dependent promoter, e.g., a U6snRNA or H1-RNA promoter, for the expression of non-coding RNAs including, but not limitedto, U snRNAs or miRNAs. In another aspect, the promoter can be a polymerase II U snRNAdependent 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 byreference herein in its entirety).

[0120] Additional promotors can be found in Wang, E. T.-S. & Poukalov, K. K. Methods andcompositions to confer regulation to gene therapy cargoes by heterologous use of alternativesplicing cassettes. World Patent (2022); Boyne, A. R., Danos, O. F., Volles, M. J. & Guo, X.61Regulation of gene expression by aptamer-mediated modulation of alternative splicing. US Patent(2022); Ranum, P. T., Monteys, A. M., Hundley, A. A. & Davidson, B. L. Compositions andmethods for inducible alternative splicing regulation of gene expression. World Patent (2021);Monteys, A. M. et al. Regulated control of gene therapies by drug-induced splicing. Nature 1-5(2021); Doshi, A., Sadeghi, F., Varadarajan, N. & Cirino, P. C. Small-molecule inducibletranscriptional control in mammalian cells. Crit. Rev. Biotechnol. 40, 1131–1150 (2020); Monteys,A. M. et al. Regulated control of gene therapies with a drug induced switch. 2020.02.21.956664(2020) doi:10.1101 / 2020.02.21.956664.; Davidson, B. L., Monteys, A. M., AI HUNDLEY, A. &Ranum, P. T. ALTERNATIVE SPLICING REGULATION OF GENE EXPRESSION ANDTHERAPEUTIC METHODS. PCT (2020); Boyne, A. R., Olivier DANOS, F., Michael VOLLES,J. & Xuecui, G. U. O. Regulation of gene expression by aptamer-mediated modulation ofalternative splicing. World Patent (2016); REGULATABLE EXPRESSION SYSTEMS. WorldPatent; BERGLUND, John, Andrew DELGADO, Elizabeth JENQUIN, Jana, Rose WANG, Eric,Tzy-Shi. GENE THERAPY VECTORS. World Patent, incorporated herein by reference in theirentirety.Further Capsid Modifications

[0121] In an embodiment, the viral capsid protein may comprise one or more mutationsrelative to wild type. In an example embodiment, the one or more mutations comprise a K449Rsubstitution in a capsid polypeptide of AAV 9 20507, or a substitution in an analogous position ofa capsid polypeptide from AAV9, AAV9 K449R (or K449R AAV9), AAV1, AAVrhlO, AAVDJ, AAV-DJ8, AAV5, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP.A), AAVG2B-26, AAVG2B13, AAVTH1.1-32, AAVTH1.1- 35, AAV-PHP.B2 (PHP.B2), AAV-PHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.B-ATP, AAV-PHP.BATT-T, AAV-PHP.B- DGT-T, AAV-PHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP,AAV-PHP.B-QQP, AAV-PHP.B-SNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.BNQT, AAV-PHP.B- EGS, AAV-PHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAVPHP.B-DST, AAV-PHP.B-STP, AAV-PHP.B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP,AAV-PHP.В- TMР, AAV-PHP.В-ТТР, АAV-PHP.S / G2A12, AA VG2A 15 / G2A3 (G2A3),AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b,AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8,AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84,62AAV9.9, AAV 10, AAV11, AAV 12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42- lb,AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8,AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12,AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5,AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVI-7 / rh.48, AAVI8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.5 1, AAV3. l / hu.6,AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55,AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.1O, AAV16. 12 / hu.11, AAV29.3 / bb.1,AAV29.5 / bb.2, AAV106.1 / hu.37, AAV1 14.3 / hu.40, AAVI27.2 / hu.41, AAV127.5 / hu.42,AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145. l / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55,AAV161.10 / hu.60, AAVI61.6 / hu.6l, AAV33.12 / hu. 17, AAV33.4 / hu.15, AAV33.8 / hu. 16,AAV52 / hu.19, AAV52. l / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7,AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68,AAVrh.70, AAVpi. 1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55,AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-l / hu.l, AAVH-5 / hu.3,AAVLG- 10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN72l-8 / rh.43, AAVCh.5,AAVCh.5RI, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5RI, AAVCy.5R2,AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu. 1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5,AAVhu.6, AAVhu.7, AAVhu.9, AAVhu. 10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16,AAVhu.1 7, AAVhu. 18, AAVhu.20, AAVhu.21, AAVhu.22, AA Vhu.23.2, AAVhu.24,AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32,AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43,AAVhu.44, AAVhu.44RI, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47,AAVhu.48, AAVhu.48RI, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52,AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.6l, AAVhu.63,AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu .t 19, AAVrh.2, AAVrh.2R, AAVrh.8,AAVrh.8R, AAVrh.1O, AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18,AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31,AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38,AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48. L2, AAVrh.48.2,AAVrh.49, AAVA.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58,63AAVA.61, AAVrh.64, AAVA.64R1, AAVA.64R2, AAVrh.67, AAVrh.73, AAVrh.74,AAVA8R, AAVA8R A586R mutant, AAVA8R R533A mutant, AAAV, BAAV, caprine AAV,bovine AAV, AAVhEl . 1, AAVhErl.5, AAVhERI . 14, AAVhErl .8, AAVhErl. 16, AAVhErl. 18,AAVhErl .35, AAVhErl 7, AAVhErl .36, AAVhEr2.29, AAVhEr2.4, AAVhEr2. 16,AAVhEr2.30, AAVhEr2.3 1, AAVhEr2.36, AAVhERI.23, AAVhEr3. 1, AAV2.5T, AAV-PAEC,AAV-LK01, AAV-LK02, AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07,AAV-LK08, AAV-LK09, AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14,AAV-LK15, AAV-LK16, AAV- LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4,AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC 11, AAV-РAEC 12, AAV-2-premiRNA-IOI, AAV-8h, AAV- 8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAVShuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8,AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu. 19,AAVhu. 11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22,AAV54.7 / hu.24, AAV54. 1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPEN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7. 1,AAV CBr-7. 10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7,AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3,AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAVCHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6. 1, AAV CHt-6. 10, AAV CHt-6.5, AAV CHt-6.6,AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAVCHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-1O, AAV CKd-2, AAV CKd-3, AAV CKd-4,AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B 1, AAV CKd-B2, AAV CKd-B3, AAVCKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-HI, AAVCKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAVCKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLgF5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-l, AAV CLvl-1, AAV Clvl-1O, AAVCLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-l 3, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8,AAV Clvl-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-DI,AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAVCLv-D8, AAV CLv-El, AAV CLv-KI, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv64L5, AAV CLv-L6, AAV CLv-MI, AAV CLv-MI 1, AAV CLv-M2, AAV CLv-M5, AAV CLvM6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-RI, AAV CLv-R2, AAV CLv-R3,AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, АAV CLv-R9, AAVCSp-1, AAV CSp-1O, AAV CSp-l 1, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAVCSp-7, AAV CSp-8, AAV CSp-8. 10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355,AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13,AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2,AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8,and / or AAVF9 / HSC9 and variants thereof. In an example embodiment, the K449R substitutedAAV capsid is selected from SEQ ID NO: 20507.

[0122] In an embodiment, the viral capsid protein may comprise additional targeting motifsthat are in addition to the transferrin receptor binding modification of the present disclosure.Without being bound by theory the additional targeting moieties can be antibodies or fragmentsthereof. In an embodiment, the additional targeting moiety can be any molecule or compositioncapable of recognizing, binding, attaching to, or otherwise interacting with a binding partner thatcan 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 ormolecules that are present on the surface of a target cell. In an embodiment, the binding partner isunique to a cell type or cell state or a to a group of related cell types or cell states. In anembodiment, the binding partner is a receptor, channel, or other complex present on the surface ofa target cell. These additional targeting moieties can be used to target, e.g., specific cell types orcell states within those the set of target cells targeted by the transferrin receptor bindingmodification. As used herein, "cell state" is used to describe transient elements of a cell's identity.Cell state can be thought of as the transient characteristic profile or phenotype of a cell. Cell statesarise transiently during time-dependent processes, either in a temporal progression that isunidirectional (e.g., during differentiation, or following an environmental stimulus) or in a statevacillation 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 oror circadian rhythm) or can transitionbetween 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 type65(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.

[0123] In an embodiment, the additional targeting moiety is or includes a peptide or apolypeptide. In an embodiment, the additional targeting moiety is or includes an antibody orfragment thereof. Exemplary antibodies and fragments thereof are described in greater detailelsewhere herein, see e.g., discussion on exemplary cargos. In an embodiment, the additionaltargeting moiety is or includes an aptamer. In an embodiment, the additional targeting moiety isor includes a small molecule. In an embodiment, the additional targeting moiety is or includes anucleic acid (e.g., DNA or RNA). In an embodiment, the additional targeting moiety is or includesa receptor. In an embodiment, the additional targeting moiety is or includes a receptor ligand. Inan embodiment, the additional targeting moiety is or includes a carbohydrate (e.g., a sugar). In anembodiment, the additional targeting moiety is or includes a lipid. In an embodiment, theadditional targeting moiety is an engineered protein scaffold. In an embodiment, the additionaltargeting moiety is an affibody. In an embodiment, the additional targeting moiety is an antibodymimetic. In an embodiment, the additional targeting moiety is an engineered binding protein, suchas a designed ankyrin repeat proteins (DARPins) (see e.g., Plückthun et al., Annu. Rev. Pharmacol.Toxicol. (2015) 55(1): 489-511), avimers (Silverman et al., Nat. Biotechnol. (2005) 23 (12): 1556-1561 and Jeong et al. Nat. Biotechnol. (2005) 23(12): 1493-1494), or affibodies (see e.g., Nord etal., Nat. Biotechnol. (1997) 15(8):772-777). In an embodiment, the additional targeting moiety isa receptor ligand or binding protein. In an embodiment, the additional targeting moiety is attachedor otherwise coupled to the capsid surface. In an embodiment, the additional targeting moiety isencoded by a vector that produces a capsid of the present invention described herein.

[0124] In an embodiment, the capsid polypeptide can be covalently modified by the covalentcoupling of at least one compound comprising a lactam moiety (e.g., B-lactam) to at least oneamino group of an amino acid residue of the capsid of the AAV vectors (see, for example, thepublished International PCT application No. PCT / EP2021 / 080832 and U.S. Patent No.US11382988, the contents of which are incorporated by reference herein in their entireties). Inother embodiments, a ligand, e.g., an 7-mer covalently linked to a primary amino group of a capsidpolypeptide via a CSNH- bond, (see, e.g., U.S. Patent No. 11,648,319, the content of which isincorporated by reference herein in its entirety).66

[0125] In an embodiment, the transferrin receptor binding modification can be bound to anAAV capsid polypeptide through a specific protein: protein binding pair that forms a covalent,e.g., isopeptide, bond. For example, the SpyTag / SpyCatcher bioconjugation technology can beused to append a targeting ligand, e.g., the transferrin receptor binding modification, to the surfaceof a capsid protein, where it can specifically bind a transferrin receptor, expressed on the cell ofinterest (see, for example, the published U.S. Patent Application No. 2020 / 0140492, the contentof which is incorporated by reference herein in its entirety). According to this embodiment, thetransferrin receptor binding modification can be fused in frame to the 13 amino acid SpyTagpeptide. Advantages of this approach include binding of the SpyTag - transferrin receptor bindingmodification to a fully assembled AAV capsid and the relative ease of testing different 7-mertransferrin receptor binding modifications using the same AAV preparation. The SpyCatchermoiety can be bound to the AAV capsid polypeptide either covalently or non-covalently, forexample, using a protein binding domain-specific for one or more epitopes on the surface of thecapsid polypeptide.Engineered Vectors and Vector Systems

[0126] Also provided herein are vectors and vector systems that can encode one or more ofthe engineered polypeptides described herein that includes one or more of the transferrin receptorbinding modifications of the present invention, including but not limited to engineered viralpolynucleotides (e.g., polynucleotides encoding engineered AAV capsid proteins). In a preferredembodiment, provided herein is a vector system comprising one or more vectors encoding atransferrin receptor binding modification effective to increase transduction of central nervoussystem tissues (CNS), optionally further comprising a vector encoding a recombinant viral genomecomprising a transgene. In preferred embodiment, the transferrin receptor binding modificationencoded in the vector system binds to Transferrin Receptor (TFR1). As used in this context,engineered viral capsid polynucleotides refers to any one or more of the polynucleotides describedherein encoding an engineered viral capsid as described elsewhere herein and / or polynucleotide(s)encoding one or more engineered viral capsid proteins described elsewhere herein. Further, wherethe vector includes an engineered viral capsid polynucleotide described herein, the vector can alsobe referred to and considered an engineered vector or system thereof although not specificallynoted as such. In embodiments, the vector can contain one or more polynucleotides encoding oneor more elements of an engineered viral capsid described herein. The vectors and systems thereof67can be useful in producing bacterial, fungal, yeast, plant cells, animal cells, and transgenic animalsthat can express one or more components of the engineered viral capsid, particle, or othercompositions described herein. Within the scope of this disclosure are vectors containing one ormore of the polynucleotide sequences described herein. One or more of the polynucleotides thatare part of the engineered viral capsid and system thereof described herein can be included in avector or vector system.

[0127] In an embodiment, a vector used in the production of the rAAVs disclosed hereincomprises a rep gene and cap gene). The rep gene typically encodes Rep78, Rep68, Rep52 andRep40 from a single ORF. These replication factors aid AAV genome replication and virionassembly. 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 bytranscription from a start codon (ACG) and alternative splicing. The cap gene also encodes, froman in-frameshifted ORF, an assembly-activating protein (AAP). The AAP is essential for capsidassembly.

[0128] In an embodiment, the vector can include an engineered viral (e.g., AAV) capsidpolynucleotide having a 3' polyadenylation signal. In an embodiment, the 3' polyadenylation is anSV40 polyadenylation signal. In an embodiment the vector does not have splice regulatoryelements. In an embodiment, the vector includes one or more minimal splice regulatory elements.In an embodiment, the vector can further include a modified splice regulatory element, whereinthe modification inactivates the splice regulatory element. In an embodiment, the modified spliceregulatory element is a polynucleotide sequence sufficient to induce splicing, between a rep proteinpolynucleotide and the engineered viral (e.g., AAV) capsid protein variant polynucleotide. In anembodiment, the polynucleotide sequence can be sufficient to induce splicing is a splice acceptoror a splice donor. In an embodiment, the viral (e.g., AAV) capsid polynucleotide is an engineeredviral (e.g., AAV) capsid polynucleotide as described elsewhere herein. It some embodiments, thevector does not include one or more minimal splice regulatory elements, modified splice regulatoryagent, splice acceptor, and / or splice donor.

[0129] The vectors and / or vector systems can be used, for example, to express one or more ofthe engineered viral (e.g., AAV) capsid and / or other polynucleotides in a cell, such as a producercell, 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 other68composition containing an n-mer motif of the present invention described elsewhere herein. Otheruses for the vectors and vector systems described herein are also within the scope of this disclosure.In general, and throughout this specification, the term is a tool that allows or facilitates the transferof an entity from one environment to another. In some contexts which will be appreciated by thoseof ordinary skill in the art, "vector" can be a term of art to refer to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. A vector can be a replicon, suchas a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. Generally, a vector is capable of replication whenassociated with the proper control elements.

[0130] 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 morefree ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both;and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," whichrefers to a circular double stranded DNA loop into which additional DNA segments can beinserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector,wherein virally derived DNA or RNA sequences are present in the vector for packaging into avirus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defectiveadenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterialorigin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomalmammalian vectors) are integrated into the genome of a host cell upon introduction into the hostcell, and thereby are replicated along with the host genome. Moreover, certain vectors are capableof directing the expression of genes to which they are operatively linked. Such vectors are referredto herein as "expression vectors." Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

[0131] Recombinant expression vectors can be composed of a nucleic acid (e.g., apolynucleotide) 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 operativelylinked to the nucleic acid sequence to be expressed. Within a recombinant expression vector,69"operably linked" and "operatively-linked" are used interchangeably herein and further definedelsewhere herein. In the context of a vector, the term "operably linked" is intended to mean thatthe nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allowsfor expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or ina host cell when the vector is introduced into the host cell).

[0132] In an embodiment, the vector can be a bicistronic vector. In an embodiment,bicistronic vector can be used for one or more elements of the engineered viral (e.g., AAV) capsidsystem described herein. In an embodiment, expression of elements of the engineered viral (e.g.,AAV) capsid system described herein can be driven by a suitable constitutive or tissue specificpromoter. Where the element of the engineered viral (e.g., AAV) capsid system is an RNA, itsexpression can be driven by a Pol III promoter, such as a U6 promoter. In an embodiment, the twoare combined.Cell-based Vector Amplification and Expression

[0133] 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 transferrin receptor bindingmodification of the present disclosure described herein (e.g., nucleic acid transcripts, proteins,enzymes, and combinations thereof) in a suitable host cell.

[0134] In an embodiment, the suitable host cell is a prokaryotic cell. Suitable host cellsinclude, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. Thevectors can be viral-based or non-viral based. In an embodiment, the suitable host cell is аeukaryotic cell. In an embodiment, the suitable host cell is a suitable bacterial cell. Suitablebacterial cells include, but are not limited to, bacterial cells from the bacteria of the speciesEscherichia coli. Many suitable strains of E. coli are known in the art for expression of vectors.These include, but are not limited to Pir1, Stb12, Stb13, Stbl4, TOP10, XL1 Blue, and XL10 Gold.In an embodiment, the host cell is a suitable insect cell. Suitable insect cells include those fromSpodoptera frugiperda. Suitable strains of S. frugiperda cells include, but are not limited to, Sf9and Sf21. In an embodiment, the host cell is a suitable yeast cell. In an embodiment, the yeast cellcan be from Saccharomyces cerevisiae. In an embodiment, the host cell is a suitable mammaliancell. Many types of mammalian cells have been developed to express vectors. Suitable mammaliancells include, but are not limited to, HEK293, Chinese Hamster Ovary Cells (CHOs), mousemyeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO70Rb50, HepG G2, DIKХ-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryofibroblasts (CEFs). Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990).

[0135] In an embodiment, the vector can be a yeast expression vector. Examples of vectors forexpression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMВО 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 (InVitrogenCorp, San Diego, Calif.). As used herein, a "yeast expression vector" refers to a nucleic acid thatcontains one or more sequences encoding an RNA and / or polypeptide and may further contain anydesired elements that control the expression of the nucleic acid(s), as well as any elements thatenable the replication and maintenance of the expression vector inside the yeast cell. Many suitableyeast expression vectors and features thereof are known in the art; for example, various vectorsand techniques are illustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press,New York, 2007) and Buckholz, R.G. and Gleeson, M.A. (1991) Biotechnology (NY) 9(11): 1067-72. Yeast vectors can contain, without limitation, a centromeric (CEN) sequence, an autonomousreplication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linkedto a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an originof replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids, yeast artificialchromosomes, 2μ plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors,and episomal plasmids.

[0136] In an embodiment, the vector is a baculovirus vector or expression vector and can besuitable for expression of polynucleotides and / or proteins in insect cells. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series(Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers,1989. Virology 170: 31-39). rAAV (recombinant Adeno-associated viral) vectors are preferablyproduced in insect cells, e.g., Spodoptera frugiperda Sf9 insect cells, grown in serum-freesuspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g.,Sigma Aldrich (EX-CELL 405).71

[0137] In an embodiment, the vector is a mammalian expression vector. In an embodiment,the mammalian expression vector is capable of expressing one or more polynucleotides and / orpolypeptides in a mammalian cell. Examples of mammalian expression vectors include, but arenot limited to, pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBOJ. 6: 187-195). The mammalian expression vector can include one or more suitable regulatoryelements capable of controlling expression of the one or more polynucleotides and / or proteins inthe mammalian cell. For example, commonly used promoters are derived from polyoma,adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art.More detail on suitable regulatory elements is described elsewhere herein.

[0138] For other suitable expression vectors and vector systems for both prokaryotic andeukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

[0139] In an embodiment, the recombinant mammalian expression vector is capable ofdirecting expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specificpromoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cellreceptors (Winoto and Baltimore, 1989. EMBО J. 8: 729-733) and immunoglobulins (Baneiji, etal., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specificpromoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), andmammary gland-specific promoters (e.g., milk whey promoter, U.S. Pat. No. 4,873,316 andEuropean Application Publication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) andthe a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With regardsto these prokaryotic and eukaryotic vectors, mention is made of U.S. Patent 6,750,059, the contentsof which are incorporated by reference herein in their entirety. Other embodiments can utilize viralvectors, with regards to which mention is made of U.S. Patent application 13 / 092,085, the contentsof which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements72are known in the art and in this regard, mention is made of U.S. Patent 7,776,321, the contents ofwhich are incorporated by reference herein in their entirety. In an embodiment, a regulatoryelement can be operably linked to a transgene in a recombinant genome packaged by theengineered AAV capsid system so as to drive expression of the one or more elements of thetransgene delivered by the viral vector as described herein in a tissue specific manner.

[0140] Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In anembodiment, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryoticcell or as an intermediate vector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packaging system). In an embodiment, aprokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as toprovide a source of one or more proteins for delivery to a host cell or host organism.

[0141] In an embodiment, the vector can be a fusion vector or fusion expression vector. In anembodiment, fusion vectors add a number of amino acids to a protein encoded therein, such as tothe amino terminus, carboxy terminus, or both of a recombinant protein. Such fusion vectors canserve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification. In an embodiment, expression ofpolynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carriedout in Escherichia coli with vectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion polynucleotides and / or proteins. In an embodiment, thefusion expression vector can include a proteolytic cleavage site, which can be introduced at thejunction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotideor protein to enable separation of the recombinant polynucleotide or protein from the fusion vectorbackbone or other fusion moiety subsequent to purification of the fusion polynucleotide or protein.Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smithand Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E bindingprotein, or protein A, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-73315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0142] In an embodiment, one or more vectors driving expression of one or more elements ofan engineered viral (e.g., AAV) capsid system or other composition containing a transferrinreceptor binding modification described herein are introduced into a host cell such that expressionof the elements of the engineered delivery system described herein direct formation of anengineered viral (e.g., AAV) capsid system or other composition containing a transferrin receptorbinding modification described herein (including but not limited to an engineered gene transferagent particle, which is described in greater detail elsewhere herein). For example, differentelements of the engineered viral (e.g., AAV) capsid system or other composition containing atransferrin receptor binding modification described herein can each be operably linked to separateregulatory elements on separate vectors. RNA(s) of different elements of the engineered deliverysystem described herein can be delivered to an animal or mammal or cell thereof to produce ananimal or mammal or cell thereof that constitutively or inducibly or conditionally expressesdifferent elements of the engineered viral (e.g., AAV) capsid system or other compositioncontaining a transferrin receptor binding modification described herein that incorporates one ormore elements of the engineered viral (e.g., AAV) capsid system or other composition containinga transferrin receptor binding modification described herein or contains one or more cells thatincorporates and / or expresses one or more elements of the engineered viral (e.g., AAV) capsidsystem or other composition containing a transferrin receptor binding modification describedherein.

[0143] In an embodiment, two or more of the elements expressed from the same or differentregulatory element(s) can be combined in a single vector, with one or more additional vectorsproviding any components of the system not included in the first vector. Engineeredpolynucleotides of the present invention that are combined in a single vector may be arranged inany suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' withrespect to ("downstream" of) a second element. The coding sequence of one element may belocated on the same or opposite strand of the coding sequence of a second element, and orientedin the same or opposite direction. In an embodiment, a single promoter drives expression of atranscript encoding one or more engineered viral (e.g., AAV) capsid proteins or other compositioncontaining a transferrin receptor binding modification described herein, embedded within one or74more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all ina single intron). In an embodiment, the engineered polynucleotides of the present invention(including but not limited to engineered viral polynucleotides) can be operably linked to andexpressed from the same promoter.Vector Features

[0144] The vectors can include additional features that can confer one or more functionalitiesto the vector, the polynucleotide to be delivered, a virus particle produced there from, orpolypeptide expressed thereof. Such features include, but are not limited to, regulatory elements,selectable markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, and thelike. It will be appreciated by those skilled in the art that the design of the expression vector andadditional features included can depend on such factors as the choice of the host cell to betransformed, the level of expression desired, etc.Regulatory Elements

[0145] In embodiments, the polynucleotides and / or vectors thereof described herein(including, but not limited to, the engineered AAV capsid polynucleotides of the presentdisclosure) can include one or more regulatory elements that can be operatively linked to thepolynucleotide. The term "regulatory element" is intended to include promoters, enhancers,internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcriptiontermination signals, such as polyadenylation signals and poly-U sequences). Such regulatoryelements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatoryelements include those that direct constitutive expression of a nucleotide sequence in many typesof host cell and those that direct expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). A tissue-specific promoter can direct expressionprimarily 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 directexpression in a temporal-dependent manner, such as in a cell-cycle dependent or developmentalstage-dependent manner, which may or may not also be tissue or cell-type specific. In anembodiment, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol IIIpromoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or morepol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples75of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol IIpromoters 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 theCMV enhancer) (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, thedihydrofolate reductase promoter, the ẞ-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EFla promoter. Also encompassed by the term "regulatory element" areenhancer 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 exons2 and 3 of rabbit ẞ-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).

[0146] In an embodiment, the regulatory sequence can be a regulatory sequence described inU.S. Pat. No. 7,776,321, U.S. Pat. Pub. No. 2011 / 0027239, and PCT publication WO2011 / 028929, the contents of which are incorporated by reference herein in their entirety. In anembodiment, the vector can contain a minimal promoter. In an embodiment, the minimal promoteris the Mecp2 promoter, tRNA promoter, or U6. In a further embodiment, the minimal promoteris tissue specific. In an embodiment, the length of the vector polynucleotide the minimal promotersand polynucleotide sequences is less than 4.4Kb.

[0147] To express a polynucleotide, the vector can include one or more transcriptional and / ortranslational initiation regulatory sequences, e.g., promoters, that direct the transcription of thegene and / or translation of the encoded protein in a cell. In an embodiment a constitutive promotermay be employed. Suitable constitutive promoters for mammalian cells are generally known inthe art and include, but are not limited to SV40, CAG, CMV, EF-1a, ẞ-actin, RSV, and PGK.Suitable constitutive promoters for bacterial cells, yeast cells, and fungal cells are generally knownin the art, such as a T-7 promoter for bacterial expression and an alcohol dehydrogenase promoterfor expression in yeast.

[0148] In an embodiment, the regulatory element can be a regulated promoter. "Regulatedpromoter" refers to promoters that direct gene expression not constitutively, but in a temporallyand / or spatially-regulated manner, and includes tissue-specific, tissue-preferred and induciblepromoters. In an embodiment, the regulated promoter is a tissue specific promoter as previouslydiscussed elsewhere herein. Regulated promoters include conditional promoters and induciblepromoters. In an embodiment, conditional promoters can be employed to direct expression of apolynucleotide in a specific cell type, under certain environmental conditions, and / or during a76specific state of development. Suitable tissue specific promoters can include, but are not limitedto, liver specific promoters (e.g., APOA2, SERPIN A1 (hAAT), СYР3A4, and MIR122),pancreatic cell promoters (e.g., INS, IRS2, Pdx1, Alx3, Ppy), cardiac specific promoters (e.g.Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTnl), NPPA (ANF), Slc8a1 (Ncx1)), centralnervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3beta)), skin cell specific promoters (e.g., FLG, K14, TGM3), immune cell specific promoters, (e.g.ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cellspecific promoters (e.g., Pbsn, Upk2, Sbp, Fer114), endothelial cell specific promoters (e.g., ENG),pluripotent and embryonic germ layer cell specific promoters (e.g. Oct4, NANOG, Synthetic Oct4,T brachyury, NES, SOX17, FOXA2, MIR122), and muscle cell specific promoter (e.g., Desmin).Other tissue and / or cell specific promoters are discussed elsewhere herein and can be generallyknown in the art and are within the scope of this disclosure.

[0149] Inducible / conditional promoters can be positively inducible / conditional promoters(e.g., a promoter that activates transcription of the polynucleotide upon appropriate interactionwith 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 arepressor) until the repressor condition of the promotor is removed (e.g., inducer binds a repressorbound to the promoter stimulating release of the promoter by the repressor or removal of achemical repressor from the promoter environment). The inducer can be a compound,environmental condition, or other stimulus. Thus, inducible / conditional promoters can beresponsive 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 limitedto, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn,XVE / OlexA, GVG, and pOp / LhGR.

[0150] In an embodiment, the vector or system thereof can include one or more elementscapable 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 ororganelle. Such organelles can include, but are not limited to, nucleus, ribosome, endoplasmicreticulum, Golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasmamembrane, cell wall, peroxisome, centrioles, etc.77CpG content

[0151] In one aspect, a rAAV vector, including an rAAV vector genome as described herein,comprises at least one synthetic AAV ITR, wherein one or more CpG islands (a cytosine basefollowed immediately by a guanine base (a CpG) in which the cytosines in such arrangement tendto be methylated) that typically occur at, or near the transcription start site in an ITR are deletedand / or substituted. In one aspect, deletion, or reduction in the number of CpG islands can reducethe immunogenicity of the rAAV vector. This results from a reduction or complete inhibition inTLR-9 binding to the rAAV vector DNA sequence, which occurs at CpG islands. It is also wellknown that methylation of CpG motifs results in transcriptional silencing. Removal of CpG motifsin the ITR is expected to result in decreased TLR-9 recognition and / or decreased methylation andtherefore decreased transgene silencing. In some aspects, it is the minimal functional ITR in whichone or more CpG islands are deleted and / or substituted. In one aspect, AAV ITR2 is known tocontain 16 CpG islands of which one or more, or all 16 can be deleted.

[0152] In some aspects, at least 1 CpG motif is deleted and / or substituted, e.g., at least 4 ormore 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 CpGmotifs. The phrase "deleted and / or substituted" as used herein means that one or both nucleotidesin the CpG motif is deleted, substituted with a different nucleotide, or any combination of deletionsand substitutions.

[0153] In certain aspects, the transgene nucleic acid sequence can also be codon optimized toenhance expression in vivo and / or to reduce the number of CpG islands and avoid an innateimmune response to the vector. An example of CpG depletion can be found in the publishedInternational Application No. PCT / US2023 / 067901, the content of which is incorporated byreference herein in its entirety.Dual AAV vectors

[0154] Various strategies have been investigated to overcome the limitation of AAV cargocapacity. Several groups have attempted to "force" large genes into one of the many AAV capsidsavailable by developing the so-called oversize vectors. Although administration of oversize AAVvectors can achieve therapeutically relevant levels of transgene expression in rodent and caninemodels of human inherited diseases, including the retina of the Abca4~ / ~ and shaker 1 (shl) mousemodels of STGD and USHIB, the mechanism underlying oversize AAV-mediated transductionremains elusive. Oversize AAV vectors do not contain a pure population of intact large size78genomes but rather a heterogeneous mixture of mostly truncated genomes<5 kb in length.Following infection, reassembly of these truncated genomes in the target cell nucleus has beenproposed as a mechanism for oversize AAV vector transduction. Independent of transductionmechanism and in vivo efficacy, the heterogeneity in oversize AAV genome sizes is a majorlimitation for their application in human gene therapy.

[0155] Alternatively, the inherent ability of AAV genomes to undergo intermolecularconcatemerization can be exploited to transfer large genes in vivo by splitting a large geneexpression cassette into halves (<5 kb in size), each contained in one of two separate (dual) AAVvectors. In the dual AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3' endof the 5' - half vector and a splice acceptor (SA) signal is placed at the 5' end of the 3-half vector.Upon coinfection of the same cell by the dual .AAV vectors and inverted terminal repeat (ITR)-mediated head-to-tail concatemerization of the two halves, trans-splicing results in the productionof a mature mRNA and full-size protein. Trans-splicing has been successfully used to express largegenes in muscle and retina.

[0156] Alternatively, the two halves of a large transgene expression cassette contained in dualAAV vectors may contain homologous overlapping sequences (at the 3' end of the 5 -half vectorand at the 5' end of the 3 '-half vector, dual AAV overlapping), which will mediate reconstitutionof a single large genome by homologous recombination. This strategy depends on therecombinogenic properties of the transgene overlapping sequences.

[0157] A third dual AAV strategy (hybrid) is based on adding a highly recombinogenic regionfrom an exogenous gene [i.e. alkaline phosphatase, AP] to the trans-splicing vector. The addedregion is placed downstream of the SD signal in the 5'-half vector and upstream of the SA signalin the 3 -half vector in order to increase recombination between the dual AAVs. The published USpatent application No. 2010 / 003218 and US Patent No. 10,494,645, both of which are incorporatedby reference herein in their entireties, provide additional examples of dual vector systems.Selectable Markers and Tags

[0158] One or more of the engineered polynucleotides of the present disclosure (e.g., anengineered viral (e.g., AAV) capsid polynucleotide) can be operably linked, fused to, or otherwisemodified to include a polynucleotide that encodes or is a selectable marker or tag, which can be apolynucleotide or polypeptide. In an embodiment, the polypeptide encoding a polypeptideselectable marker can be incorporated in the engineered polynucleotide of the present disclosure79(e.g., an engineered viral (e.g., AAV) capsid polynucleotide) such that the selectable markerpolypeptide, when translated, is inserted between two amino acids between the N- and C- terminusof an engineered polypeptide (e.g., the engineered AAV capsid polypeptide) or at the N- and / or Cterminus of the engineered polypeptide (e.g., an engineered AAV capsid polypeptide). In anembodiment, the selectable marker or tag is a polynucleotide barcode or unique molecularidentifier (UMI).

[0159] It will be appreciated that the polynucleotide encoding such selectable markers or tagscan be incorporated into a polynucleotide encoding one or more components of the engineeredAAV capsid system described herein in an appropriate manner to allow expression of theselectable marker or tag. Such techniques and methods are described elsewhere herein and will beinstantly appreciated by one of ordinary skill in the art in view of this disclosure. Many suchselectable markers and tags are generally known in the art and are intended to be within the scopeof this disclosure.

[0160] Suitable selectable markers and tags include, but are not limited to, affinity tags, suchas 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 specificenzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such asreaction with FlAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that containrestriction enzyme or other enzyme cleavage sites; DNA segments that encode products thatprovide resistance against otherwise toxic compounds including antibiotics, such as,spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II(NEO), hygromycin phosphotransferase (HPT) and the like; DNA and / or RNA segments thatencode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophicmarkers); DNA and / or RNA segments that encode products which can be readily identified (e.g.,phenotypic markers such as ẞ-galactosidase, GUS; fluorescent proteins such as green fluorescentprotein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins);polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition oftwo DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon bya restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP,80FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecularidentifier (UMI), DNA sequences required for a specific modification (e.g., methylation) thatallows its identification. Other suitable markers will be appreciated by those of skill in the art.

[0161] Selectable markers and tags can be operably linked to one or more components of theengineered AAV capsid system or other compositions and / or systems described herein via suitablelinker, such as a glycine or glycine serine linkers as short as GS or GG up to (GGGGG)3 (SEQ IDNO: 1733) or (GGGGS)3 (SEQ ID NO: 1734). Other suitable linkers are described elsewhereherein.

[0162] The vector or vector system can include one or more polynucleotides encoding one ormore transferrin receptor binding modifications. In an embodiment, the transferrin receptorbinding modification encoding polynucleotides can be included in the vector or vector system,such as a viral vector system, such that they are expressed within and / or on the virus particle(s)produced such that the virus particles can be targeted to specific cells, tissues, organs, etc. In anembodiment, the transferrin receptor binding modification encoding polynucleotides can beincluded in the vector or vector system such that the engineered polynucleotide(s) of the presentdisclosure (e.g., an engineered viral (e.g., AAV) capsid polynucleotide(s)) and / or productsexpressed therefrom include the transferrin receptor binding modification and can be targeted tospecific cells, tissues, organs, etc. In an embodiment, such as non-viral carriers, the transferrinreceptor binding modification can be attached to the carrier (e.g., polymer, lipid, inorganicmolecule etc.) and can be capable of targeting the carrier and any attached or associated engineeredpolynucleotide(s) of the present disclosure, the engineered polypeptides, or other compositions ofthe present disclosure described herein, to specific cells, tissues, organs, etc. In an embodiment,the specific cells are CNS cells.Cell-free Vector and Polynucleotide Expression

[0163] In an embodiment, the polynucleotide(s) encoding a transferrin receptor bindingmodification of the present disclosure can be expressed from a vector or suitable polynucleotidein a cell-free in vitro system. In an embodiment, the polynucleotide encoding one or more featuresof the engineered AAV capsid system can be expressed from a vector or suitable polynucleotidein a cell-free in vitro system. In other words, the polynucleotide can be transcribed and optionallytranslated in vitro. In vitro transcription / translation systems and appropriate vectors are generallyknown in the art and commercially available. Generally, in vitro transcription and in vitro81translation systems replicate the processes of RNA and protein synthesis, respectively, outside ofthe cellular environment. Vectors and suitable polynucleotides for in vitro transcription can includeT7, SP6, T3, promoter regulatory sequences that can be recognized and acted upon by anappropriate polymerase to transcribe the polynucleotide or vector.

[0164] In vitro translation can be stand-alone (e.g., translation of a purifiedpolyribonucleotide) or linked / coupled to transcription. In an embodiment, the cell-free (or in vitro)translation system can include extracts from rabbit reticulocytes, wheat germ, and / or E. coli. Theextracts can include various macromolecular components that are needed for translation ofexogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA, synthetases, initiation,elongation factors, termination factors, etc.). Other components can be included or added duringthe translation reaction, including but not limited to, amino acids, energy sources (ATP, GTP),energy regenerating systems (creatine phosphate and creatine phosphokinase (eukaryoticsystems)) (phosphoenol pyruvate and pyruvate kinase for bacterial systems), and other co-factors(Mg2+, K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNAstarting 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 DNAtemplate as a starting material (e.g., E coli-based systems). In these systems transcription andtranslation 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 arecommercially available.Codon Optimization of Vector Polynucleotides

[0165] As described elsewhere herein, the polynucleotide encoding a transferrin receptorbinding modification of the present disclosure and / or other polynucleotides described herein, orthe transgene contained within the recombinant AAV genome can be codon optimized. In anembodiment, polynucleotides of the engineered AAV capsid system described herein can be codonoptimized. In an embodiment, one or more polynucleotides contained in a vector ("vectorpolynucleotides") described herein that are in addition to an optionally codon optimizedpolynucleotide encoding a transferrin receptor binding modification, including but not limited to,embodiments of the engineered AAV capsid system described herein, can be codon optimized. Ingeneral, codon optimization refers to a process of modifying a nucleic acid sequence for enhancedexpression in the host cells of interest by replacing at least one codon (e.g., about or more than82about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that aremore frequently or most frequently used in the genes of that host cell while maintaining the nativeamino acid sequence. Various species exhibit particular bias for certain codons of a particularamino acid. Codon bias (differences in codon usage between organisms) often correlates with theefficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependenton, among other things, the properties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell isgenerally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the "Codon Usage Database" availableat 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 theyear 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are also available, such as Gene Forge(Aptagen; Jacobus, PA), are also available. In an embodiment, one or more codons (e.g., 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA / RNA-targeting Casprotein corresponds to the most frequently used codon for a particular amino acid. As to codonusage in yeast, reference is made to the online Yeast Genome database available atwww.yeastgenome.org / community / codon_usage.shtml, or Codon selection in yeast, Bennetzenand Hall, J Biol Chem. 1982 Mar 25;257(6):3026-31. As to codon usage in plants including algae,reference is made to Codon usage in higher plants, green algae, and cyanobacteria, Campbell andGowri, 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 andcyanelle genes in different plant and algal lineages, Morton BR, J Mol Evol. 1998 Apr;46(4):449-59.

[0166] The vector polynucleotide can be codon optimized for expression in a specific celltype, tissue type, organ type, and / or subject type. In an embodiment, a codon optimized sequenceis a sequence optimized for expression in a eukaryote, e.g., humans (i.e., being optimized forexpression in a human or human cell), or for another eukaryote, such as another animal (e.g., amammal or avian) as is described elsewhere herein. Such codon optimized sequences are withinthe ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the83polynucleotide is codon optimized for a specific cell type. Such cell types can include, but are notlimited to, epithelial cells (including skin cells, cells lining the gastrointestinal tract, cells liningother hollow organs), nerve cells (nerves, brain cells, spinal column cells, nerve support cells (e.g.,astrocytes, glial cells, Schwann cells etc.), muscle cells (e.g., cardiac muscle, smooth muscle cells,and skeletal muscle cells), connective tissue cells (fat and other soft tissue padding cells, bonecells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immunesystem cells, germ cells, and combinations thereof. Such codon optimized sequences are withinthe ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, thepolynucleotide is codon optimized for a specific tissue type. Such tissue types can include, but arenot limited to, muscle tissue, connective tissue, nervous tissue, and epithelial tissue. Such codonoptimized sequences are within the ambit of the ordinary skilled artisan in view of the descriptionherein. In an embodiment, the polynucleotide is codon optimized for a specific organ. Such organsinclude, but are not limited to, muscles, skin, intestines, liver, spleen, brain, lungs, stomach, heart,kidneys, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinationsthereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan inview of the description herein.

[0167] In an embodiment, a vector polynucleotide is codon optimized for expressionparticular cells, such as prokaryotic or eukaryotic cells. The eukaryotic cells may be those of orderived from a particular organism, such as a plant or a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as discussed herein, e.g., mouse, rat, rabbit,dog, livestock, or non-human mammal or primate.inNon-Viral Vectors and Carriers

[0168] In an embodiment, the vector is a non-viral vector or carrier. In an embodiment, nonviral vectors can have the advantage(s) of reduced toxicity and / or immunogenicity and / orincreased bio-safety as compared to viral vectors The terms of art "Non-viral vectors and carriers"and as used herein in this context refers to molecules and / or compositions that are not based onone or more component of a virus or virus genome (excluding any nucleotide to be delivered and / orexpressed 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 AAVcapsid polynucleotide) or other composition of the present disclosure described herein and can becapable of ferrying the polynucleotide to a cell and / or expressing the polynucleotide. It will be84appreciated that this does not exclude the inclusion of a virus-based polynucleotide that is to bedelivered. For example, if a gRNA to be delivered is directed against a virus component and it isinserted or otherwise coupled to an otherwise non-viral vector or carrier, this would not make saidvector a "viral vector". Non-viral vectors and carriers include naked polynucleotides, chemicalbased carriers, polynucleotide (non-viral) based vectors, and particle-based carriers. It will beappreciated that the term "vector" as used in the context of non-viral vectors and carriers refers topolynucleotide vectors and "carriers" used in this context refers to a non-nucleic acid orpolynucleotide molecule or composition that be attached to or otherwise interact with apolynucleotide to be delivered, such as an engineered AAV capsid polynucleotide of the presentdisclosure.Naked Polynucleotides

[0169] In an embodiment one or more engineered AAV capsid polynucleotides or otherpolynucleotides of the present disclosure described elsewhere herein can be included in a nakedpolynucleotide. The term of art "naked polynucleotide" as used herein refers to polynucleotidesthat are not associated with another molecule (e.g., proteins, lipids, and / or other molecules) thatcan often help protect it from environmental factors and / or degradation. As used herein, associatedwith includes, but is not limited to, linked to, adhered to, adsorbed to, enclosed in, enclosed in orwithin, mixed with, and the like. Naked polynucleotides that include one or more of the engineeredAAV capsid polynucleotides or other polynucleotides of the present disclosure described hereincan be delivered directly to a host cell and optionally expressed therein. The naked polynucleotidescan have any suitable two- and three-dimensional configurations. By way of non-limitingexamples, naked polynucleotides can be single-stranded molecules, double stranded molecules,circular molecules (e.g., plasmids and artificial chromosomes), molecules that contain portionsthat are single stranded and portions that are double stranded (e.g., ribozymes), and the like. In anembodiment, the naked polynucleotide contains only the engineered AAV capsidpolynucleotide(s) or other polynucleotides of the present disclosure. In an embodiment, the nakedpolynucleotide can contain other nucleic acids and / or polynucleotides in addition to the engineeredAAV capsid polynucleotide(s) or other polynucleotides of the present disclosure describedelsewhere herein. The naked polynucleotides can include one or more elements of a transposonsystem. Transposons and system thereof are described in greater detail elsewhere herein.85Non-Viral Polynucleotide Vectors

[0170] In an embodiment, one or more ofthe engineered AAV capsid polynucleotides or otherpolynucleotides 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 andvector 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-linearvector) vectors, Ministrings, mini-intronic plasmids, PSK systems (post-segregationally killingsystems), ORT (operator repressor titration) plasmids, and the like. See e.g., Hardee et al. 2017.Genes. 8(2):65.

[0171] In an embodiment, the non-viral polynucleotide vector can have a conditional origin ofreplication. In an embodiment, the non-viral polynucleotide vector can be an ORT plasmid. In anembodiment, the non-viral polynucleotide vector can have a minimalistic immunologicallydefined gene expression. In an embodiment, the non-viral polynucleotide vector can have one ormore post-segregationally killing system genes. In an embodiment, the non-viral polynucleotidevector is AR-free. In an embodiment, the non-viral polynucleotide vector is a minivector. In anembodiment, the non-viral polynucleotide vector includes a nuclear localization signal. In anembodiment, the non-viral polynucleotide vector can include one or more CpG motifs. In anembodiment, the non-viral polynucleotide vectors can include one or more scaffold / matrixattachment 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 presentdisclosure. S / MARs are AT-rich sequences that play a role in the spatial organization ofchromosomes through DNA loop base attachment to the nuclear matrix. S / MARs are often foundclose to regulatory elements such as promoters, enhancers, and origins of DNA replication.Inclusion of one or S / MARs can facilitate a once-per-cell-cycle replication to maintain the nonviral polynucleotide vector as an episome in daughter cells. In an embodiment, the S / MARsequence is located downstream of an actively transcribed polynucleotide (e.g., one or moreengineered AAV capsid polynucleotides or other polynucleotides or molecules of the presentdisclosure) included in the non-viral polynucleotide vector. In an embodiment, the S / MAR can bea S / MAR from the beta-interferon gene cluster. See e.g., Verghese et al. 2014. Nucleic Acid Res.8642:e53; Xu et al. 2016. Sci. China Life Sci. 59:1024-1033; Jin et al. 2016. 8:702-711; Koirala etal. 2014. Adv. Exp. Med. Biol. 801:703-709; and Nehlsen et al. 2006. Gene Ther. Mol. Biol.10:233-244, whose techniques and vectors can be adapted for use in the present disclosure.

[0172] In an embodiment, the non-viral vector is a transposon vector or system thereof. Asused herein, "transposon" (also referred to as transposable element) refers to a polynucleotidesequence that is capable of moving form location in a genome to another. There are several classesof transposons. Transposons include retrotransposons and DNA transposons. Retrotransposonsrequire the transcription of the polynucleotide that is moved (or transposed) in order to transposethe polynucleotide to a new genome or polynucleotide. DNA transposons are those that do notrequire reverse transcription of the polynucleotide that is moved (or transposed) in order totranspose the polynucleotide to a new genome or polynucleotide. In an embodiment, the non-viralpolynucleotide vector can be a retrotransposon vector. In an embodiment, the retrotransposonvector includes long terminal repeats. In an embodiment, the retrotransposon vector does notinclude long terminal repeats. In an embodiment, the non-viral polynucleotide vector can be aDNA transposon vector. DNA transposon vectors can include a polynucleotide sequence encodinga transposase. In an embodiment, the transposon vector is configured as a non-autonomoustransposon vector, meaning that the transposition does not occur spontaneously on its own. In someofthese embodiments, the transposon vector lacks one or more polynucleotide sequences encodingproteins required for transposition. In an embodiment, the non-autonomous transposon vectorslack one or more Ac elements.

[0173] In an embodiment, a non-viral polynucleotide transposon vector system can include afirst polynucleotide vector that contains the engineered AAV capsid polynucleotide(s) or otherpolynucleotides, 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 thatincludes a polynucleotide capable of encoding a transposase coupled to a promoter to driveexpression of the transposase. When both are expressed in the same cell the transposase can beexpressed from the second vector and can transpose the material between the TIRs on the firstvector (e.g., the engineered AAV capsid polynucleotide(s) or other polynucleotides or moleculesof the present disclosure) and integrate it into one or more positions in the host cell's genome. Inan embodiment the transposon vector or system thereof can be configured as a gene trap. In anembodiment, the TIRs can be configured to flank a strong splice acceptor site followed by a87reporter and / or other gene (e.g., one or more of the engineered AAV capsid polynucleotide(s) orother polynucleotides or molecules of the present disclosure) and a strong poly A tail. Whentransposition occurs while using this vector or system thereof, the transposon can insert into anintron of a gene and the inserted reporter or other gene can provoke a mis-splicing process and asa result it in activates the trapped gene.

[0174] Any suitable transposon system can be used. Suitable transposon and systems thereofcan include Sleeping Beauty transposon system (Tc1 / mariner superfamily) (see e.g., Ivics 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), FrogPrince (Tc1 / mariner superfamily) (see e.g., Miskey et al. 2003 Nucleic Acid Res. 31(23):6873-6881) and variants thereof.Chemical Carriers

[0175] In an embodiment, the engineered AAV capsid polynucleotide(s) or otherpolynucleotides or other molecules of the present disclosure described herein can be coupled to achemical carrier. Chemical carriers that can be suitable for delivery of polynucleotides can bebroadly classified into the following classes: (i) inorganic particles, (ii) lipid-based, (iii) polymerbased, and (iv) peptide based. They can be categorized as (1) those that can form condensedcomplexes with a polynucleotide (such as the engineered AAV capsid polynucleotide(s) of thepresent disclosure), (2) those capable of targeting specific cells, (3) those capable of increasingdelivery of the polynucleotide or other molecules (such as the engineered AAV capsidpolynucleotide(s) )of the present disclosure to the nucleus or cytosol of a host cell, (4) thosecapable of disintegrating from DNA / RNA in the cytosol of a host cell, and (5) those capable ofsustained or controlled release. It will be appreciated that any one given chemical carrier caninclude features from multiple categories. The term "particle" as used herein, refers to any suitablesized particles for delivery of the compositions (including particles, polypeptides, polynucleotides,and other compositions described herein) present disclosure described herein. Suitable sizesinclude macro-, micro-, and nano-sized particles.

[0176] In an embodiment, the non-viral carrier can be an inorganic particle. In an embodiment,the inorganic particle, can be a nanoparticle. The inorganic particles can be configured andoptimized by varying size, shape, and / or porosity. In an embodiment, the inorganic particles areoptimized to escape from the reticulo endothelial system. In an embodiment, the inorganic particles88can be optimized to protect an entrapped molecule from degradation. The suitable inorganicparticles 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 combinationsthereof), magnetic compounds, particles, and materials, (e.g., supermagnetic iron oxide andmagnetite), quantum dots, fullerenes (e.g., carbon nanoparticles, nanotubes, nanostrings, and thelike), and combinations thereof. Other suitable inorganic non-viral carriers are discussed elsewhereherein.

[0177] In an embodiment, the non-viral carrier can be lipid-based. Suitable lipid-based carriersare also described in greater detail herein. In an embodiment, the lipid-based carrier includes acationic lipid or an amphiphilic lipid that is capable of binding or otherwise interacting with anegative charge on the polynucleotide to be delivered (e.g., such as an engineered AAV capsidpolynucleotide of the present disclosure). In an embodiment, chemical non-viral carrier systemscan include a polynucleotide (such as the engineered AAV capsid polynucleotide(s)) or othercomposition or molecule of the present disclosure) and a lipid (such as a cationic lipid). These arealso referred to in the art as lipoplexes. Other embodiments of lipoplexes are described elsewhereherein. In an embodiment, the non-viral lipid-based carrier can be a lipid nano emulsion. Lipidnano emulsions can be formed by the dispersion of an immiscible liquid in another stabilizedemulsifying 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 capsidpolynucleotide(s) of the present disclosure). In an embodiment, the lipid-based non-viral carriercan be a solid lipid particle or nanoparticle.

[0178] In an embodiment, the non-viral carrier can be peptide-based. In an embodiment, thepeptide-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. Inan embodiment, peptide carriers can be used in conjunction with other types of carriers (e.g.,polymer-based carriers and lipid-based carriers to functionalize these carriers). In an embodiment,the functionalization is targeting a host cell. Suitable polymers that can be included in the polymerbased 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 engineered89AAV capsid polynucleotides of the present disclosure), polymethacrylate, and combinationsthereof.

[0179] In an embodiment, the non-viral carrier can be configured to release an engineereddelivery system polynucleotide that is associated with or attached to the non-viral carrier inresponse to an external stimulus, such as pH, temperature, osmolarity, concentration of a specificmolecule or composition (e.g., calcium, NaCl, and the like), pressure and the like. In anembodiment, the non-viral carrier can be a particle that is configured includes one or more of theengineered AAV capsid polynucleotides or other compositions of the present disclosure describeherein and an environmental triggering agent response element, and optionally a triggering agent.In an embodiment, the particle can include a polymer that can be selected from the group ofpolymethacrylates and polyacrylates. In an embodiment, the non-viral particle can include one ormore embodiments of the compositions microparticles described in US Pat. Pubs. 20150232883and 20050123596, whose techniques and compositions can be adapted for use in the presentdisclosure.

[0180] In an embodiment, the non-viral carrier can be a polymer-based carrier. In anembodiment, the polymer is cationic or is predominantly cationic such that it can interact in acharge-dependent manner with the negatively charged polynucleotide to be delivered (such as theengineered AAV capsid polynucleotide(s) of the present disclosure). Polymer-based systems aredescribed in greater detail elsewhere herein.Viral Vectors

[0181] In an embodiment, the vector is a viral vector. The term of art "viral vector" and asused herein in this context refers to polynucleotide based vectors that contain one or more elementsfrom or based upon one or more elements of a virus that can be capable of expressing andpackaging a polynucleotide, such as an engineered AAV capsid polynucleotide, cargo, or othercomposition or molecule of the present disclosure, into a virus particle and producing said virusparticle when used alone or with one or more other viral vectors (such as in a viral vector system).Viral vectors and systems thereof can be used for producing viral particles for delivery of and / orexpression and / or generation of one or more compositions of the present disclosure describedherein (including, but not limited to, any viral particle and associated cargo). The viral vector canbe part of a viral vector system involving multiple vectors. In an embodiment, systemsincorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors90can include adenoviral-based vectors, adeno associated vectors, helper-dependent adenoviral(HdAd) vectors, hybrid adenoviral vectors, and the like. Other embodiments of viral vectors andviral particles produce therefrom are described elsewhere herein. In an embodiment, the viralvectors are configured to produce replication incompetent viral particles for improved safety ofthese systems.Adenoviral vectors, Helper-dependent Adenoviral vectors, and Hybrid Adenoviral Vectors

[0182] In an embodiment, the vector can be an adenoviral vector. In an embodiment, theadenoviral vector can include elements such that the virus particle produced using the vector orsystem thereof can be serotype 2, 5, or 9. In an embodiment, the polynucleotide to be deliveredvia the adenoviral particle can be up to about 8 kb. Thus, in an embodiment, an adenoviral vectorcan include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb toabout 8 kb. Adenoviral vectors have been used successfully in several contexts (see e.g., Teramatoet al. 2000. Lancet. 355:1911-1912; Lai et al. 2002. DNA Cell. Biol. 21:895-913; Flotte et al.,1996. Hum. Gene. Ther. 7:1145-1159; and Kay et al. 2000. Nat. Genet. 24:257-261. The vectorcan encode the engineered AAV capsids, said capsids forming adenoviral particles.

[0183] In an embodiment the vector can be a helper-dependent adenoviral vector or systemthereof. These are also referred to in the field as "gutless" or "gutted" vectors and are a modifiedgeneration of adenoviral vectors (see e.g., Thrasher et al. 2006. Nature. 443:E5-7). In anembodiment of the helper-dependent adenoviral vector system one vector (the helper) can containall the viral genes required for replication but contains a conditional gene defect in the packagingdomain. The second vector of the system can contain only the ends of the viral genome, one ormore engineered AAV capsid polynucleotides, and the native packaging recognition signal, whichcan 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 genedelivery in several contexts (see e.g., Simonelli et al. 2010. J Am Soc Gene Ther. 18:643-650;Cideciyan et al. 2009. N Engl J Med. 361:725-727; Crane et al. 2012. Gene Ther. 19(4):443-452;Alba et al. 2005. Gene Ther. 12:18-S27; Croyle et al. 2005. Gene Ther. 12:579-587; Amalfitanoet al. 1998. J. Virol. 72:926-933; and Morral et al. 1999. PNAS. 96:12816-12821). The techniquesand vectors described in these publications can be adapted for inclusion and delivery of theengineered AAV capsid polynucleotides described herein. In an embodiment, the polynucleotideto be delivered via the viral particle produced from a helper-dependent adenoviral vector or system91thereof can be up to about 38 kb. Thus, in an embodiment, an adenoviral vector can include a DNApolynucleotide 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).

[0184] In an embodiment, the vector is a hybrid-adenoviral vector or system thereof. Hybridadenoviral vectors are composed of the high transduction efficiency of a gene-deleted adenoviralvector and the long-term genome-integrating potential of adeno-associated, and transposon basedgene transfer. In an embodiment, such hybrid vector systems can result in stable transduction andlimited integration site. See e.g., Balague et al. 2000. Blood. 95:820-828; Morral et al. 1998. Hum.Gene Ther. 9:2709-2716; Kubo and Mitani. 2003. J. Virol. 77(5): 2964-2971; Zhang et al. 2013.PloS One. 8(10) e76771; and Cooney et al. 2015. Mol. Ther. 23(4):667-674), whose techniquesand vectors described therein can be modified and adapted for use in the engineered AAV capsidsystem of the present disclosure. In an embodiment, a hybrid-adenoviral vector can include one ormore features of a retrovirus and / or an adeno-associated virus. In an embodiment the hybridadenoviral vector can include one or more features of a spuma retrovirus or foamy virus (FV). Seеe.g., Ehrhardt et al. 2007. Mol. Ther. 15:146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841,whose techniques and vectors described therein can be modified and adapted for use in theengineered AAV capsid system ofthe present disclosure. Advantages of using one or more featuresfrom the FVs in the hybrid-adenoviral vector or system thereof can include the ability of the viralparticles produced therefrom to infect a broad range of cells, a large packaging capacity ascompared to other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See alsoe.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 theengineered AAV capsid system of the present disclosure.Adeno Associated Vectors

[0185] In an embodiment, the engineered vector or system thereof can be an adeno-associatedvector (AAV). See, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351(1994). Although similar to adenoviral vectors in some of their features, AAVs have somedeficiency in their replication and / or pathogenicity and thus can be safer than adenoviral vectors.In an embodiment, the AAV can integrate into a specific site on chromosome 19 of a human cellwith no observable side effects. In an embodiment, the capacity of the AAV vector, system thereof,92and / or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can includeone or more engineered capsid polynucleotides described herein.

[0186] The AAV vector or system thereof can be operably linked to a regulatory sequence,said regulatory sequence encoding one or more regulatory molecules. In an embodiment theregulatory molecules can be promoters, enhancers, repressors and the like, which are described ingreater detail elsewhere herein. In an embodiment, the AAV vector or system thereof can includeone or more polynucleotides that can encode one or more regulatory proteins. In an embodiment,the promoter can be a tissue specific promoter as previously discussed. In an embodiment, thetissue specific promoter can drive expression of an engineered capsid AAV capsid polynucleotidedescribed herein

[0187] The AAV vector or system thereof can include one or more polynucleotides that canencode one or more capsid proteins, such as the engineered AAV capsid proteins describedelsewhere 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.

[0188] In an embodiment, the AAV vector or system thereof can include one or moreadenovirus helper factors or polynucleotides that can encode one or more adenovirus helperfactors. Such adenovirus helper factors can include, but are not limited, E1A, E1B, E2A, E4ORF6,and VA RNAs. In an embodiment, a producing host cell line expresses one or more of theadenovirus helper factors.

[0189] The AAV vector or system thereof can be configured to produce AAV particles havinga specific serotype. In an embodiment, the serotype can be AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV8, AAV9 or any combinations thereof. In an embodiment, the AAV can be AAV1,AAV2, AAV5, AAV9 or any combination thereof. One can select the AAV of the AAV withregard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5, 9 or a hybrid capsidAAVI, AAV2, AAV5, AAV9 or any combination thereof for targeting brain and / or neuronal cells;and one can select AAV4 for targeting cardiac tissue; and one can select AAV8 for delivery to theliver. Thus, in an embodiment, an AAV vector or system thereof capable of producing AAVparticles capable of targeting the brain and / or neuronal cells can be configured to generate AAVparticles having serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combinationthereof. In an embodiment, an AAV vector or system thereof capable of producing AAV particles93capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV4serotype. In an embodiment, an AAV vector or system thereof capable of producing AAV particlescapable 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.

[0190] The nucleotide sequences of the genomes of the AAV serotypes are known in the art.For example, the complete genome of AAV1 is provided in GenBank Accession No. NC 002077;the complete genome of AAV2 is provided in GenBank Accession No. NC 001401 and Srivastavaet al., J. Virol., 45: 555-564 (1983); the complete genome of AAV3 is provided in GenBankAccession 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 completegenome of AAV6 is provided in GenBank Accession No. NC 00 1862; at least portions of AAV7 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); tlieAAV10 genome is provided in Mol. Then, 13(1): 67-76 (2006); the AAV11 genome is providedin Virology, 330(2): 375-383 (2004); AAV PHP.B is described by Deverman et al., NatureBiotech. 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.

[0191] It will be appreciated that while the different serotypes can provide some level of cell,tissue, and / or organ specificity, each serotype still is multi-tropic and thus can result in tissuetoxicity if using that serotype to target a tissue that the serotype is less efficient in transducing.Thus, in addition to achieving some tissue targeting capacity via selecting an AAV of a particularserotype, it will be appreciated that the tropism of the AAV serotype can be modified by anengineered AAV capsid described herein. As described elsewhere herein, variants of wild-typeAAV of any serotype can be generated via a method described herein and determined to have aparticular cell-specific tropism, which can be the same or different as that of the reference wildtype AAV serotype. In an embodiment, the cell, tissue, and / or specificity ofthe wild-type serotypecan be enhanced (e.g., made more selective or specific for a particular cell type that the serotypeis already biased towards). For example, wild-type AAV9 is biased towards muscle and brain inhumans (see e.g., Srivastava. 2017. Curr. Opin. Virol. 21:75-80.) By including an engineered AAVcapsid and / or capsid protein variant of wild-type AAV9 as described herein, the bias for e.g., brain94can be reduced or eliminated and / or the septicity increased such that the brain specificity appearsreduced in comparison, thus enhancing the specificity for the muscle as compared to the wild-typeAAV9. As previously mentioned, inclusion of an engineered capsid and / or capsid protein variantof a wild-type AAV serotype can have a different tropism than the wild-type reference AAVserotype. For example, an engineered AAV capsid and / or capsid protein variant of AAV9 can havespecificity for a tissue other than muscle or brain in humans.

[0192] In an embodiment, the AAV vector is a hybrid AAV vector or system thereof. HybridAAVs are AAVs that include genomes with elements from one serotype that are packaged into acapsid derived from at least one different serotype. For example, if it is the rAAV2 / 5 that is to beproduced, and if the production method is based on the helper-free, transient transfection methoddiscussed below, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the sameas discussed for rAAV2 production. However, the 2nd plasmid, the pRepCap will be different. Inthis plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV2, while the Cap gene isderived from AAV5. The production scheme is the same as the above-mentioned approach forAAV2 production. The resulting rAAV is called rAAV2 / 5, in which the genome is based onrecombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropismdisplayed by this AAV2 / 5 hybrid virus should be the same as that of AAV5. It will be appreciatedthat wild-type hybrid AAV particles suffer the same specificity issues as with the non-hybrid wildtype serotypes previously discussed.

[0193] Advantages achieved by the wild-type based hybrid AAV systems can be combinedwith the increased and customizable cell-specificity that can be achieved with the engineered AAVcapsids can be combined by generating a hybrid AAV that can include an engineered AAV capsiddescribed elsewhere herein. It will be appreciated that hybrid AAVs can contain an engineeredAAV capsid containing a genome with elements from a different serotype than the reference wildtype serotype that the engineered AAV capsid is a variant of. For example, a hybrid AAV can beproduced that includes an engineered AAV capsid that is a variant of an AAV9 serotype that isused to package a genome that contains components (e.g., AAV2 ITRs) from an AAV2 serotype.As with wild-type based hybrid AAVs previously discussed, the tropism of the resulting AAVparticle will be that of the engineered AAV capsid.95

[0194] A tabulation of certain wild-type AAV serotypes as to these cells can be found inGrimm, D. et al, J. Virol. 82: 5887-5911 (2008) reproduced below as Table E. Further tropismdetails can be found in Srivastava. 2017. Curr. Opin. Virol. 21:75-80 as previously discussed.Table ECell Line AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV8 AAV9Huh-7 13 100 2.5 0.0 0.1 10 0.7 0.0HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1HeLa 3 100 2.0 0.1 6.7 1 0.2 0.1HepG2 3 100 16.7 0.3 1.7 5 0.3 NDHep1A 20 100 0.2 1.0 0.1 1 0.2 0.0911 17 100 11 0.2 0.1 17 0.1 NDCHO 100 100 14 1.4 333 50 10 1.0COS 33 100 33 3.3 5.0 14 2.0 0.5MeWo 10 100 20 0.3 6.7 10 1.0 0.2NIH3T3 10 100 2.9 2.9 0.3 10 0.3 NDA549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1Monocytes 1111 100 ND ND 125 1429 ND NDImmature DC 2500 100 ND ND 222 2857 ND NDMature DC 2222 100 ND ND 333 3333 ND ND

[0195] In an embodiment, the AAV vector or system thereof is AAV rh.74 or AAV rh.10.

[0196] In an embodiment, the AAV vector or system thereof is configured as a "gutless"vector, similar to that described in connection with a retroviral vector. In an embodiment, the"gutless" AAV vector or system thereof can have the cis-acting viral DNA elements involved ingenome amplification and packaging in linkage with the heterologous sequences of interest (e.g.,a transgene encoding a therapeutic protein or nucleic acid of interest)).96Table F: Example virus sequences - see Figure 17 for an example alignment of the examplevirus sequences.SEQIDSequence Name1 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGONQQTLKFSVAGPSNMAVOGRNYIPGPSYRQORVSTTVTONNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTTPVPADPPTPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*AAV92 AAV9K449RMAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGL VLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDOOLKAGDNPYLKYNHADAEFOERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSOWLGDRVITTSTRTWALPTYNNHLYKOISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*3 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRPKRLNFKLFNIOVKEVTTNDGAAVIVTTIANNLTSTVOQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGD VHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL4 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADS VPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTAAV297567IANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNLMAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKGAVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGGGAPMAIADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKOKQISSQSGASNDNHYYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLE KLFNIQVRGVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTGTVNHQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNLMAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKOPARKRLNFGOTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLOGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPOIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNLMTDGYLPDWLEDNLSEGVREWWALOPGAPKPKANOQHODNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQQRLQGDTSFGGNLGRAVFQAKKRVLEPLGL VEQAGETAPGKKRPLIESPQQPDSSTGIGKKGKOPAKKKLVFEDETGAGDGPPEGSTSGAMSDDSEMRAAAGGAAVEGGOGADGVGNASGDWHCDSTWSEGHVTTTSTRTWVLPTYNNHLYKRLGESLOSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNWGMRPKAMRVKIFNIQVKEVTTSNGETTVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGLVTGNTSQQQTDRNAFYCLEYFPSQMLRTGNNFEITYSFEKVPFHSMYAHSQSLDRLMNPLIDQYLWGLQSTTTGTTLNAGTATTNFTKLRPTNFSNFKKNWLPGPSIKOOGFSKTANONYKIPATGSDSLIKYETHSTLDGRWSALTPGPPMATAGPADSKFSNSQLIFAGPKQNGNTATVPGTLIFTSEEELAATNATDTDMWGNLPGGDQSNSNLPTVDRLTALGAVPGMVWQNRDIYYQGPIWAKIPHTDGHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFSSTPVNSFITQYSTGQVS VQIDWEIQKERSKRWNPEVQFTSNYGQQNSLLWAPDAAGKYTEPRAIGTRYL THHLAAV3AAV3BAAV48 MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGL VLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVAAV59891011QVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPL VDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGTGEYRTTRPIGTRYLTRPLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGL VEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATРAAVGPTTNTMASGGGAPMADNNINEGAVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNV RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADV YVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLOSSSTDPATGDVHVMGALPGMVWODRDVYLOGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGL VEEGAKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGOTGDSESVPDPQPLGEPPAAPSSVGSGTVAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLTSTIQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTGNNFEFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPSTMAEQAKNWLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSL VNPGVAMATHKDDEDRFFPSSGVLIFGKTGATNKTTLENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQVVNNQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFТРАKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNLMAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGOTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNLAAV6AAV7AAV812 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGL VLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGL VEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGLGPNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNErh.89913141516GTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQALGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLVRTQTTGTGGTQTLAFSQAGPSSMANQARNWVPGPCYRQQRVSTTTNONNNSNFAWTGAAKFKLNGRDSLMNPGVAMASHKDDDDRFFPSSGVLIFGKOGAGNDGVDYSQVLITDEEEIKATNPVATEEYGAVAINNQAANTQAQTGLVHNQGVIPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPLTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGL VEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMААAAGGGAPMADADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHHL YKQISNGTSGGSTNDDNTYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRPKPKRLNFKLFNIQVKEVTOTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVVFMIPQYGYLTLNNSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSONNNSNFAWTGATKYHLNGRDSL VNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEOYGVVADNLOOONAAPIVGAVNSOGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFOAKKRVLEPLGL VEEGAKTAPGKKRPLESPOEPDSSSGIGKKGKOPAKKRLNFEEDTGAGDGPPEGSDTSAMSSDIEMRAAPGGNAVDAGOGSDGVGNASGDWHCDSTWSEGKVTTTSTRTWVLPTYNNHLYLRLGTTSNSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNNWGLRPKAMRVKIFNIQVKEVTTSNGETTVANNLTSTVQIFADSSYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGIVTGENQNQTDRNAFYCLEYFPSQMLRTGNNFEMAYNFEKVPFHSMYAHSQSLDRLMNPLLDQYL WHLQSTTSGETLNQGNAATTFGKIRSGDFAFYRKNWLPGPCVKQQRFSKTASQNYKIPASGGNALLKYDTHYTLNNRWSNIAPGPPMATAGPSDGDFSNAQLIFPGPSVTGNTTTSANNLLFTSEEEIAATNPRDTDMFGQIADNNQNATTAPITGNVTAMGVLPGMVWONRDIYYQGPIWAKIPHADGHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFTAARVDSFITQYSTGQVAVQIEWEIEKERSKRWNPEVQFTSNYGNQSSMLWAPDTTGKYTEPRVIGSRYLTNHLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGL VEEAAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGESESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTONEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTQGTQQLLFSQAGPANMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSL VNPGVAMATHKDDEERFFPSSGVLMFGKQGAGRDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQANTGPIVGNVNSQGALPGMVWONRDVYLOGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNLMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGL VLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGL VEEGAKTAPGKKRPLESPQEPDSSSGIGKKGKQPARKRLNFEEDTGAGDGPPEGSDTSAMSSDIEMRAAPGGNAVDAGQGSDGVGNASGDWHCDSTWSEGKVTTTSTRTWVLPTYNNHLYLRLGTTSSSNTYNGFSTPWGYFDFNRFHCHFSPRD WQRLINNNWGLRPKAMRVKIFNIQVKEVTTSNGETTVANNLTS100rh.10rh.74AAV10AAV11TVQIFADSSYELPYVMDAGQEGSLPPFPNDVFMVPQYGYCGIVTGENQNQTDRNAFYCLEYFPSQMLRTGNNFEMAYNFEKVPFHSMYAHSQSLDRLMNPLLDQYLWHLQSTTSGETLNQGNAATTFGKIRSGDFAFYRKNWLPGPCVKQQRFSKTASQNYKIPASGGNALLKYDTHYTLNNRWSNIAPGPPMATAGPSDGDFSNAQLIFPGPSVTGNTTTSANNLLFTSEEEIAATNPRDTDMFGQIADNNQNATTAPITGNVTAMGGVLPGMVWQNRDIYYQGPIWAKIPHADGHFHPSPLIGGFGLKHPPPQIFIKNTPVPANPATTFTAARVRVDSFITQYSTGQVAVQIEWEIEKERSKRWNPEVQFTSNYGNQSSMLWA WAPDTTGKYTEPVIGSRYLTNHL17 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNGRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDKQLEQGDNPYLKYNHADAEFQQRLATDTSFGGNLGRAVFQAKKRILEPLGL VEEGVKTAPGKKRPLEKTPNRPTNPDSGKAPAKKKQKDGEPADSARRTLDFEDSGAGDGPPEGSSSGEMSHDADAEMRAAPGGNAVEAGQGADGVGNASGDWHCDSTWSEGRVTTTSTRTWV TTSTRTWVLPTYNNHLYLRIGTTANSNTYNGFSTPWGYFDFNRFHCHFSPRDWQRLINNN SNGETTVANNLTSTVQIFADSTYELPYVMDAGQEGSFPPFPND GSFPPFPNDVFMVPQYGYCGVVTKNQNQTDRNAFYCLEYFPSQMLRTGNNFEVSYQFEKVPFHSMYAHSQSLDRMMNPLLDQYL WHLQSTTTGNSLNOGTATTTYGKITTGDFAYYRKNWLPGACIKQQKFSKNANONYKIPASGGDALLKYDTHTTLNGRWSNMAPGPPMATAGAGDSDFSNSQLIFAGPNPSGNTTTSSNNLLFTSEEEIATTNPRDTDMFGOIADNNONATTAPHIANLDAMGIVPGMVWQNRDIYYQGPIWAKVPHTDGHFHPSPLMGGFGLKHPPPQIFIKNTPVPANPNTTFSAARINSFLTQYSTGQVAVQIDWEIQKEHSKRWNPEVQFTSNYGTQNSMAAV1218 AAV13LWAPDNAGNYHELRAIGSRFLTHHLMTDGYLPDWLEDNLSEGVREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKRPVEQSPAEPDSSSGIGKSGQQPARKRLNFGOTGDTESVPDPQPLGOPPAAPSGVGSTTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGATNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQTASGTQQSRLLFSQAGPTSMSLQAKNWLPGPCYRQQRLSKQANDNNNSNFPWTGATKYHLNGRDSLVNPGPAMASHKDDKEKFFPMHGTLIFGKEGTNANNADLENVMITDEEEIRTTNPVATEQYGTVSNNLQNSNAGPTTGTVNHQGALPGMVWQDRDVYLOGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPOIMIKNTPVPANPPTNFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL19 MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYL LK03GPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL20 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDROLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTK101AAVDJTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNLVectors Encoding the Transgene

[0197] In an embodiment, the vector encoding the transgene (also referred to as "an artificialgenome") 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 theyparticipate in vector production, induce transgene expression, and ensure continual celltransduction. Accordingly, ~96% of the AAV genome can be removed for gene therapy. Forexample, the rep and cap genes can be substituted for the expression cassette containing a promoter(such as those described herein), a therapeutic transgene (for example, IDS) and a poly(A) tailforms the essence of all AAV vectors.

[0198] In an embodiment, additional modifications may be implemented to further increasethe efficacy of the AAV. For example, the AAV ITRs may be modified to increase the expressionof the rAAV vector upon transduction, which may allow the transgene to be expressed withoutsecond-strand DNA synthesis; the promoter may be modified to increase transcription; and thecodons in the transgene may be engineered to modify mRNA production and / or translation.

[0199] In an embodiment, the ITRs are modified to overcome second-strand synthesis afterinfection. The AAV transduction rate is restricted by the synthesis of dsDNA from the singlestranded AAV genome. ITRs initiate second-strand synthesis. In an example embodiment,modified ITRs are no longer suitable substrates for the Rep68 and Rep78 proteins. As a result, theterminal resolution of replication is obviated, and specific self-complementary AAV (scAAV)replication intermediates are produced. The scAAV intermediates comprise plus and minus strandsof DNA fused by the modified ITRs encapsulated into the virion shell. Wild-type AAVs packageeither a single plus-strand or minus-strand DNA. The modified sCAAV intermediates are deliveredto the nucleus, these plus and minus strands instantaneously anneal to form dsDNA.

[0200] In an embodiment, the cis-elements are optimized for targeted delivery. The ciselements are optimized because the packaging capacity of AAVs is restricted. In an example102embodiment, small cis-elements replace long promoter sequences for the delivery of largetherapeutic transgenes (e.g., 4.4-4.5 kbs).

[0201] In an embodiment, several strategies may be used to deliver transgenes using AAVvectors. Example approach 1 takes advantage of an AAV genome concatemerized via thehomologous recombination of ITR sequences. In this approach, transgene cassettes may be splitinto 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 morefragments.

[0202] In example approach 2, truncated transgene fragments of different lengths are packagedinto different AAV virions at undefined locations on the vector genome. Either homologousrecombination of the overlapping regions of the different AAV vector genomes or annealing ofdifferent AAV vector genomes at complementary regions via single-stranded templates producesthe transgene cassette. In an embodiment, overlapping fragments may be added to the end of theindividual AAV vectors to encourage homologous recombination.

[0203] In example approach 3, a hybrid dual-vector incorporates an overlapping region withintron splice sites in the split vector transgenes. Approach 3 uses concatemerization activity ofAAV 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 increasethe expression of full functional protein.

[0204] In example approach 4, an AAV genome is cross-packaged into the capsids of otherparvoviruses thus creating chimeric vectors. In example approach 5, intein-mediated protein transsplicing is used. Intein catalyzes protein splicing thereby causing the ligation of two polypeptidesvia trans-splicing (this approach is similar to intron-mediated RNA splicing). Multiple AAVvectors are delivered to the same cells. Each of the AAV vectors encode one of the fragments oftarget proteins, the fragments are flanked by short split inteins. The full-length protein forms afterprotein trans-splicing. See e.g., Li, C., Samulski, R.J. Engineering adeno-associated virus vectorsfor gene therapy. Nat Rev Genet 21, 255-272 (2020), herein incorporated by reference.Vector Construction

[0205] The vectors described herein can be constructed using any suitable process ortechnique. In an embodiment, one or more suitable recombination and / or cloning methods or103techniques can be used to the vector(s) described herein. Suitable recombination and / or cloningtechniques and / or methods can include, but not limited to, those described in U.S. Applicationpublication No. US 2004-0171156 A1. Other suitable methods and techniques are describedelsewhere herein.

[0206] 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 beused and / or adapted for constructing an AAV or other vector described herein. AAV vectors arediscussed elsewhere herein.

[0207] In an embodiment, the vector can have one or more insertion sites, such as a restrictionendonuclease recognition sequence (also referred to as a "cloning site"). In an embodiment, oneor more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertionsites) are located upstream and / or downstream of one or more sequence elements of one or morevectors.

[0208] Delivery vehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of an engineered AAV capsid system describedherein are as used in the foregoing documents, such as International Patent Application PublicationWO 2014 / 093622 (РCT / US2013 / 074667) and are discussed in greater detail herein.Virus Particle Production from Viral VectorsAAV Particle Production

[0209] There are two main strategies for producing AAV particles from AAV vectors andsystems thereof, such as those described herein, which depend on how the adenovirus helperfactors are provided (helper v. helper free). In an embodiment, a method of producing AAVparticles from AAV vectors and systems thereof can include adenovirus infection into cell linesthat stably harbor AAV replication and capsid encoding polynucleotides along with AAV vectorcontaining the polynucleotide to be packaged and delivered by the resulting AAV particle (e.g.,the engineered AAV capsid polynucleotide(s)). In an embodiment, a method of producing AAVparticles from AAV vectors and systems thereof can be a "helper free" method, which includesco-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 therapeutic104protein or nucleic acid operably linked to a regulatory element that promotes expression in thetarget tissue) between 2 ITRs; (2) a vector that carries the AAV Rep-Cap encoding polynucleotide,including the engineered capsid protein described herein; and helper polynucleotides. One of skillin the art will appreciate various methods and variations thereof that are both helper and -helperfree and as well as the different advantages of each system.

[0210] The engineered AAV vectors and systems thereof described herein can be produced byany of these methods.Vector and Virus Particle Delivery

[0211] A vector (including non-viral carriers) described herein can be introduced into hostcells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptidesencoded 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 asfrom viral vectors and systems thereof).

[0212] AAV capsids prepared from one or more engineered AAV capsid polynucleotides canbe used to deliver a recombinant AAV genome encoding a therapeutic protein or nucleic acid ofinterest. Alternatively, adenovirus or other plasmid or viral vector types as previously described,can be used, in particular, using formulations and doses from, for example, US Patents Nos.8,454,972 (formulations, doses for adenovirus), 8,404,658 (formulations, doses for AAV) and5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publicationsregarding the clinical trials involving lentivirus, AAV and adenovirus. For examples, for AAV,the route of administration, formulation and dose can be as in US Patent No. 8,454,972 and as inclinical trials involving AAV. For Adenovirus, the route of administration, formulation and dosecan be as in US Patent No. 8,404,658 and as in clinical trials involving adenovirus.

[0213] For plasmid delivery, the route of administration, formulation and dose can be as in USPatent No 5,846,946 and as in clinical studies involving plasmids. In an embodiment, doses canbe based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can beadjusted for patients, subjects, mammals of different weight and species. Frequency ofadministration is within the ambit of the medical or veterinary practitioner (e.g., physician,veterinarian), depending on usual factors including the age, sex, general health, other conditionsof the patient or subject and the particular condition or symptoms being addressed. The viralvectors can be injected into or otherwise delivered to the tissue or cell of interest.105

[0214] In terms of in vivo delivery, AAV is advantageous over other viral vectors for a coupleof 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 ofcausing insertional mutagenesis because it does not integrate into the host genome.

[0215] The vector(s) and virus particles described herein can be delivered into a host cell invitro, in vivo, and or ex vivo. Delivery can occur by any suitable method including, but not limitedto, physical methods, chemical methods, and biological methods. Physical delivery methods arethose methods that employ physical force to counteract the membrane barrier of the cells tofacilitate intracellular delivery of the vector. Suitable physical methods include, but are not limitedto, needles (e.g., injections), ballistic polynucleotides (e.g., particle bombardment, micro projectilegene transfer, and gene gun), electroporation, sonoporation, photoporation, magnetofection,hydroporation, and mechanical massage. Chemical methods are those methods that employ achemical to elicit a change in the cells membrane permeability or other characteristic(s) to facilitateentry of the vector into the cell. For example, the environmental pH can be altered which can elicita change in the permeability of the cell membrane. Biological methods are those that rely andcapitalize on the host cell's biological processes or biological characteristics to facilitate transportof the vector (with or without a carrier) into a cell. For example, the vector and / or its carrier canstimulate an endocytosis or similar process in the cell to facilitate uptake of the vector into the cell.

[0216] Delivery of engineered AAV capsid system components (e.g., polynucleotidesencoding engineered AAV capsid and / or capsid proteins) to cells via particles. The term "particle"as used herein, refers to any suitable sized particles for delivery of the engineered AAV capsidsystem components described herein. Suitable sizes include macro-, micro-, and nano-sizedparticles. In an embodiment, any of the of the engineered AAV capsid system components (e.g.,polypeptides, polynucleotides, vectors, and combinations thereof described herein) can be attachedto, coupled to, integrated with, otherwise associated with one or more particles or componentthereof as described herein. The particles described herein can then be administered to a cell ororganism by an appropriate route and / or technique. In an embodiment, particle delivery can beselected and be advantageous for delivery of the polynucleotide or vector components. It will beappreciated that in embodiments, particle delivery can also be advantageous for other engineeredcapsid system molecules and formulations described elsewhere herein.106Engineered Virus Particles Including an Engineered Viral (e.g., AAV) Capsid

[0217] Also described herein are engineered virus particles (also referred to here andelsewhere herein as "engineered viral particles" that can contain an engineered viral capsid (e.g.,AAV capsid, referred to as "engineered AAV particles") as described in detail elsewhere herein.It will be appreciated that the engineered AAV particles can be adenovirus-based particles, helperadenovirus-based particles, AAV-based particles, or hybrid adenovirus-based particles thatcontain at least one engineered AAV capsid proteins as previously described. An engineered AAVcapsid is one that that contains one or more engineered AAV capsid proteins as are describedelsewhere herein. In an embodiment, the engineered AAV particles can include 1-60 engineeredAAV capsid proteins described herein. In an embodiment, the engineered AAV particles cancontain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In an embodiment, the engineered AAVparticles can contain 0-59 wild-type AAV capsid proteins. In an embodiment, the engineered AAVparticles 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 AAVparticles can thus include one or more transferrin receptor binding modifications as is previouslydescribed.

[0218] The engineered AAV particle can include one or more cargo polynucleotides. Cargopolynucleotides are discussed in greater detail elsewhere herein. Methods of making theengineered AAV particles from viral and non-viral vectors are described elsewhere herein.Formulations containing the engineered virus particles are described elsewhere herein.Example Cargos

[0219] The transferrin receptor binding modifications can be coupled to or otherwiseassociated with a cargo. Cargos can include any molecule that is capable of being coupled to orassociated with the transferrin receptor binding modifications described herein. Cargos caninclude, without limitation, nucleotides, oligonucleotides, polynucleotides, amino acids, peptides,polypeptides, riboproteins, lipids, sugars, pharmaceutically active agents (e.g., drugs, imaging andother diagnostic agents, and the like), chemical compounds, and combinations thereof. In anembodiment, the cargo is DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers,107ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essentialtumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics,analgesics, antispasmodics, anti-inflammatories, antihistamines, anti-infectives, radiationsensitizers, chemotherapeutics, radioactive compounds, imaging agents, and combinations thereof.In an embodiment, the cargo is a recombinant AAV genome comprising a transgene, for example,encoding a therapeutic protein or nucleic acid, operably linked to regulatory sequences that directexpression of the therapeutic protein or nucleic acid in a target tissue, flanked by AAV ITRsequences.

[0220] In an embodiment, the cargo is capable of treating or preventing a neurological diseaseor disorder, details of which are described herein.

[0221] In an embodiment, the cargo is a morpholino, a peptide-linked morpholino, an antisenseoligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeuticpolypeptide or peptide, a PPMO, one or more peptides, one or more polynucleotides encoding aCRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein, wherein the ribonucleoproteincomprises a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other gene modifyingor therapeutic RNA and / or protein, or any combination thereof.

[0222] In an embodiment, one or more transferrin receptor binding modifications describedherein is directly attached to the cargo. In an embodiment, one or more transferrin receptor bindingmodifications described herein is indirectly coupled to the cargo, such as via a linker molecule. Inan embodiment, one or more one or more transferrin receptor binding modifications describedherein is coupled to associated with a polypeptide or other particle that is coupled to, attached to,encapsulates, and / or contains a cargo.

[0223] 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 nanoscaledeposit of a homogenous or heterogeneous material. Nanoparticles may be regular or irregular inshape and may be formed from a plurality of co-deposited particles that form a compositenanoscale particle. Nanoparticles may be generally spherical in shape or have a composite shapeformed from a plurality of co-deposited generally spherical particles. Exemplary shapes for thenanoparticles include, but are not limited to, spherical, rod, elliptical, cylindrical, disc, and the like.In an embodiment, the nanoparticles have a substantially spherical shape.108Cargo Polynucleotides

[0224] Cargos are also described elsewhere herein. In an embodiment, the cargo is a cargopolynucleotide that can be packaged into an engineered viral particle and subsequently deliveredto a cell. In an embodiment, delivery is cell selective, e.g., neurons and glial cells of the centralnervous system. In an embodiment, the one or more cargo polynucleotides are part of theengineered viral (e.g., AAV) genome of the viral (e.g., AAV) system and packaged within theengineered capsid containing a transferrin receptor binding modification of the present disclosure.The cargo polynucleotides can be packaged into an engineered viral (e.g., AAV) particle, whichcan be delivered to, e.g., a cell. In an embodiment, the cargo polynucleotide can be capable ofmodifying a polynucleotide (e.g., gene or transcript) of a cell to which it is delivered. As usedherein, "gene" can refer to a hereditary unit corresponding to a sequence of DNA that occupies aspecific 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 agenome. "Gene" can refer to the specific sequence of DNA that is transcribed into an RNAtranscript that can be translated into a polypeptide or be a catalytic RNA molecule, including butnot limited to, tRNA, siRNA, piRNA, miRNA, long-non-coding RNA and shRNA.Polynucleotide, gene, transcript, etc. modification includes all genetic engineering techniquesincluding, but not limited to, gene editing as well as conventional recombinational genemodification techniques (e.g., whole or partial gene insertion, deletion, and mutagenesis (e.g.,insertional and deletional mutagenesis) techniques.

[0225] In an embodiment, the cargo molecule is a polynucleotide that is or can encode avaccine. In an embodiment, the cargo molecule is a polynucleotide encoding an antibody.RNA Interference Agents

[0226] In an embodiment, the one or more polynucleotides may encode one or more RNAinterference agents. RNA interference agents are RNA molecules capable of suppressing geneexpressions. Examples of RNA interference agentsinclude, but are not limited to, small interferingRNAs (sIRNA), microRNAs (miRNA), and short hairpin RNAs (shRNA).

[0227] In an embodiment, the interference RNA may be a siRNAs. Small interfering RNA(siRNA) molecules are capable of inhibiting target gene expression by interfering RNA. siRNAsmay be chemically synthesized, or may be obtained by in vitro transcription, or may be synthesizedin vivo in target cell. siRNAs may comprise double-stranded RNA from 15 to 40 nucleotides in109length and can contain a protuberant region 3' and / or 5' from 1 to 6 nucleotides in length. Lengthof protuberant region is independent from total length of siRNA molecule. siRNAs may act bypost-transcriptional degradation or silencing of target messenger. In some cases, the exogenouspolynucleotides encode shRNAs. In shRNAs, the antiparallel strands that form siRNA areconnected by a loop or hairpin region.

[0228] In an embodiment, the cargo polynucleotide is an RNAi molecule, antisense molecule,and / or a gene silencing oligonucleotide or a polynucleotide that encodes an RNAi molecule,antisense molecule, and / or gene silencing oligonucleotide.

[0229] As used herein, "gene silencing oligonucleotide" refers to any oligonucleotide that canalone 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 geneexpression, RNA level(s), RNA translation, RNA transcription, that can lead to a reduction oreffective loss of a protein expression and / or function of a non-coding RNA as compared to wildtype or a suitable control. This is synonymous with the phrase "gene knockdown" Reduction ingene expression, RNA level(s), RNA translation, RNA transcription, and / or protein expression canrange from about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80,79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54,53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28,27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, to 1% orless reduction. "Gene silencing oligonucleotides" include, but are not limited to, any antisenseoligonucleotide, ribozyme, any oligonucleotide (single or double stranded) used to stimulate theRNA interference (RNAi) pathway in a cell (collectively RNAi oligonucleotides), smallinterfering RNA (siRNA), microRNA, and short-hairpin RNA (shRNA). Commercially availableprograms and tools are available to design the nucleotide sequence of gene silencingoligonucleotides for a desired gene, based on the gene sequence and other information availableto one of ordinary skill in the art.

[0230] In an embodiment, a cargo polynucleotide, such as an encoding polynucleotide, isflanked by at least a retroelement polypeptide encoding polynucleotide 3' UTR or portion thereof,such as the proximal region of about 500 base pairs of the 3' UTR. In an embodiment a cargopolynucleotide, such as an encoding polynucleotide, is flanked by a (e.g., endogenous or110engineered) retroelement polypeptide (such as a retroviral gag protein or gag homolog) 5' UTR.In an embodiment a cargo polynucleotide, such as an encoding polynucleotide, is flanked by an(e.g., endogenous or engineered) retroelement polypeptide encoding polynucleotide 5' and 3'UTR. In an embodiment, the flanking retroelement polypeptide encoding polynucleotide UTR(s)are from PNMA, Arc, PEG10 or other Sushi Class polypeptide. In an embodiment, the inclusionof the 3' UTR, the 5'UTR, or both can increase packaging and / or delivery of the cargo that theyflank. These and other packaging elements are described in greater detail elsewhere herein.Gene Modification Cargo Polynucleotides

[0231] In an embodiment, the cargo molecule can be a polynucleotide or polypeptide orpolynucleotide 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 arenot limited to, CRISPR-Cas systems. Other gene modification systems, e.g., TranscriptionActivator-like Effector (TALE)- or Zinc Finger Protein (ZFP)-based transcriptional activator;repressor, or epigenomic silencer, a RNA encoding a partial gene fragment designed fortransplacing into an endogenous RNA, one or more transfer RNAs, or a component thereof, or anOMEGA system or any component thereof, Cre-Lox, morpholinos, etc. are other non-limitingexamples of gene modification systems whose one or more components can be delivered by theengineered viral (e.g., AAV) particles described herein.

[0232] In an embodiment, the cargo molecule is or encodes a gene editing system orcomponent thereof. In an embodiment, the cargo molecule is or encodes а CRISPR-Cas systemmolecule or a component thereof. In an embodiment, the cargo molecule is a polynucleotide thatencodes one or more components of a gene modification system (such as a CRISPR-Cas system).In an embodiment the cargo molecule is or encodes a gRNA. CRISPR-Cas system as used hereinis intended to encompass by Class 1 and Class 2 CRISPR-Cas systems and derivatives of CRISPRCas systems such as base editors, prime editors, and CRISPR-associated transposases (CAST)systems.

[0233] In an embodiment, the cargo molecule can be a polynucleotide or polypeptide orpolynucleotide 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 prevents111a disease, a disorder, or a symptom thereof of a neurologic disease or disorder, and / or viruses (suchas single stranded RNA viruses). In an embodiment, the cargo molecule, whether or not deliveredwith other components of the system, operates to modify the genome, epigenome, and / ortranscriptome of a cell to which it is delivered, is such that it treats or prevents a neurologicaldisease or disorder described further herein.

[0234] In an embodiment, the cargo molecule, whether or not delivered with other componentsof the system, operates to modify the genome, epigenome, and / or transcriptome of a cell to whichit 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 expressedtheir entirety herein and can be adapted for use with the present disclosure.in

[0235] In an embodiment, the cargo molecule is or encodes an antisense oligomer or RNAmolecule, such as those described in U.S. Pat. App. Pub. US20160251398, US20150267202, andUS20180216111, the contents of which are incorporated by reference as if expressed in theirentirety herein and can be adapted for use with the present disclosure.

[0236] In an embodiment, the cargo molecule can be a peptide-oligomer, conjugate asdescribed in e.g., International Patent Application Publication WO2017106304A1, the contents ofwhich are incorporated by reference as if expressed in their entirety herein and can be adapted foruse with the present disclosure.

[0237] An embodiment of the disclosure encompasses methods of modifying a genomic locusof interest to change gene expression in a cell by introducing into the cell any of the compositionsdescribed herein.

[0238] An embodiment of the disclosure is that the above elements are comprised in a singlecomposition or comprised in individual compositions. These compositions may advantageouslybe applied to a host to elicit a functional effect on the genomic level.Polypeptides

[0239] In an embodiment, the cargo molecule may one or more polypeptides or may be anucleic acid encoding a polypeptide. The polypeptide may be a full-length protein or a functionalfragment or functional domain thereof, that is a fragment or domain that maintains the desiredfunctionality of the full-length protein. As used within this section "protein" is meant to refer tofull-length proteins and functional fragments and domains thereof. A wide array of polypeptidesmay be delivered using the engineered delivery vesicles described herein, including but not limited112to, secretory proteins, immunomodulatory proteins, anti-fibrotic proteins, proteins that promotetissue regeneration and / or transplant survival functions, hormones, anti-microbial proteins, antifibrillating polypeptides, and antibodies. The one or more polypeptides may also comprisecombinations of the aforementioned example classes of polypeptides. It will be appreciated thatany of the polypeptides described herein can also be delivered via the engineered delivery vesiclesand systems described herein via delivery of the corresponding encoding polynucleotide.Antibodies

[0240] In an embodiment, the one or more polypeptides may comprise one or more antibodies.The term "antibody" is used interchangeably with the term "immunoglobulin" herein, and includesintact antibodies, fragments of antibodies, e.g., Fab, F(ab')2 fragments, scFva, and intact antibodiesand 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 toproduce antibodies with a desired trait, e.g., enhanced binding and / or reduced FcR binding). Theterm "fragment" refers to a part or portion of an antibody or antibody chain comprising feweramino acid residues than an intact or complete antibody oro antibody chain. Fragments can beobtained via chemical or enzymatic' treatment of an intact or complete antibody or antibody chain.Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab',F(ab')2, Fabc, Fd, dAb, VHH and scF and / or Fv fragments. As used herein, a preparation of antibodyprotein "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 chemicalprecursors is considered to be substantially free. When the antibody protein or biologically activeportion thereof is recombinantly produced, it is also preferably substantially free of culturemedium, 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 ormass of the protein preparation.

[0241] In an embodiment, the antibody is a fragment or portion thereof. In an exampleembodiment, the antibody is an epitope binding protein or portion thereof. The term "bindingportion" of an antibody (or "antibody portion") includes one or more complete domains, e.g., apair of complete domains, as well as fragments of an antibody that retain the ability to specificallybind to a target molecule. It has been shown that the binding function of an antibody can be113performed by fragments of a full-length antibody. Binding fragments are produced by recombinantDNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, Fv, single chains, single-chain antibodies,e.g., scFv, and single domain antibodies.

[0242] In an embodiment, the cargo or antibody is an antibody fragment or portion. In anembodiment, the cargo is an epitope binding protein. Examples of portions of antibodies orepitope-binding proteins encompassed by the present definition include: (i) the Fab fragment,having VL, CL, VH and CH1 domains; (ii) the Fab' fragment, which' is a Fab fragment having oneor more cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd fragment having VHand CH1 domains; (iv) the Fd' fragment having VH and CH1 domains and one or more cysteineresidues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domainsof a single arm of an antibody; (vi) the dAb fragment (Ward et al., 341 Nature 544 (1989)) whichconsists of a VH domain or a VL domain that binds antigen; (vii)'isolated CDR regions or isolatedCDR regions presented in a functional framework; (viii) F(ab')2 fragments which are bivalentfragments including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix)single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., 242 Science 423 (1988);and Huston et al., 85 PNAS 5879 (1988)); (x) "diabodies" with two antigen binding sites,comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL)in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; Hollinger et al., 90 PNAS6444 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-Сh1-VH-Ch1)which, together with complementary light chain oligopeptides, form a pair of "antigen bindingregions" (Zapata et al., Protein Eng. 8(10):1057-62 (1995); and U.S. Patent No. 5,641,870).

[0243] The term "antigen-binding fragment" refers to a polypeptide fragment of animmunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with theintact antibody from which they were derived) for antigen binding (i.e., specific binding). As suchthese antibodies or fragments thereof are included in the scope of the disclosure, provided that theantibody or fragment binds specifically to a target molecule.

[0244] In an embodiment, the antibody is a single-chain antibody (scFvs). The term "singlechain variable fragment", as used herein refers to a fusion protein containing the variable region(s)of the heavy (VH) and light (VL) of an immunoglobulin that are connected via a linker peptide. Thelinker peptide typically ranges from about 10 to about 25 amino acids. The linker can be flexible114and can contain one or more glycine residues for flexibility. The linker can contain one or moreserine or threonine residues to increase or modify solubility. The Vн and light (VL) can be linkedvia the linker in any order. In an embodiment, N terminus of the VH and is coupled, via a linker, Cterminus of the (VL). In an embodiment, C terminus of the VH and is coupled, via a linker, Nterminus of the (VL). In an embodiment, the scFV is a bivalent or trivalent scFvs. In anembodiment 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 (July1993). "Diabodies": small bivalent and bispecific antibody fragments". Proceedings of theNational Academy of Sciences of the United States of America. 90 (14): 6444–8; incq, S; Bosman,F; Buyse, MA; Degrieck, R; Celis, L; De Boer, M; Van Doorsselaere, V; Sablon, E (2001)."Expression and purification of monospecific and bispecific recombinant antibody fragmentsderived from antibodies that block the CD80 / CD86-CD28 costimulatory pathway". ProteinExpression and Purification. 22 (1): 11-24. doi:10.1006 / prep.2001.1417; Le Gall, F.; Kipriyanov,SM; Moldenhauer, G; Little, M (1999). “Di-, tri- and tetrameric single chain Fv antibodyfragments against human CD19: effect of valency on cell binding". FEBS Letters. 453 (1): 164-168. doi:10.1016 / S0014-5793(99)00713-9; Huston, J. S.; Levinson, D.; Mudgett-Hunter, M.; Tai,M. S.; Novotný, J.; Margolies, M. N.; Crea, R. (1988). "Protein engineering of antibody bindingsites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced inEscherichia coli". Proceedings of the National Academy of Sciences of the United States ofAmerica. 85 (16): 5879–5883; de Graaf et al., Methods Mol Biol. 2002;178:379-87. doi:10.1385 / 1-59259-240-6:379; Zhou, H.X., J Mol Biol. 2003 May 23;329(1):1-8. doi:10.1016 / s0022-2836(03)00372-3; Bird and Walker. Trends Biotechnol. 1991 Apr;9(4):132-7. doi:10.1016 / 0167-7799(91)90044-I; Wörn et al., J Mol Biol. 2001 Feb 2;305(5):989-1010. doi:10.1006 / jmbi.2000.4265.

[0245] As used herein, "heavy chain antibody," "VHH" or "single-domain antibodies"(sdAbs) refers to an antibody which is composed only of two heavy chains and lacks the two lightchains usually found in antibodies (see, e.g., Henry and MacKenzie, Antigen recognition by singledomain antibodies: structural latitudes and constraints. MAbs. 2018 Aug-Sep; 10(6): 815–826).VHH can refer to an antibody or VHH domain. Single-domain antibodies (sdAb) are also referredto as a "nanobody", which is defined herein as an antibody fragment composed of a singlemonomeric variable antibody domain. As used herein "VHH" is used interchangeably with115"nanobody." The ~12-15 kDa variable domains of these antibodies (VHHs and VNARs) can beproduced recombinantly and can recognize antigen in the absence of the remainder of the antibodyheavy chain. In common antibodies, the antigen binding region consists of the variable domainsof the heavy and light chains (VH and VL). Heavy-chain antibodies can bind antigens despitehaving only VH domains. In an embodiment, the heavy chain antibody is an antibody derived fromcartilaginous fishes (immunoglobulin new antigen receptor (IgNAR)) or camelid ungulates. Nonlimiting examples of camelids include dromedaries, camels, llamas and alpacas.

[0246] It is intended that the term "antibody" encompass any Ig class or any Ig subclass (e.g.,the IgG1, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans andnon-human primates, and in rodents, lagomorphs, caprines, bovines, equines, ovines, etc.).

[0247] The term "Ig class" or "immunoglobulin class", as used herein, refers to the five classesof immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD,and IgE. The term "Ig subclass" refers to the two subclasses of IgM (H and L), three subclasses ofIgA (IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4)that have been identified in humans and higher mammals. The antibodies can exist in monomericor polymeric form; for example, IgM antibodies exist in pentameric f-rm, and IgA antibodies existin monomeric, dimeric or multimeric form.

[0248] The term "IgG subclass" refers to the four subclasses of immunoglobulin class IgGIgG1, IgG2, IgG3, and IgG4 that have "been identified in humans and higher mammals by theheavy chains of the immunoglobulins, V1 - y4, respectively. The term "single-chainimmunoglobulin" or "single-chain antibody" (used interchangeably herein) refers to a proteinhaving a two-polypeptide chain structure consisting of a heavy and a light chain, said chains beingstabilized, for example, by interchain peptide linkers, which has the ability to specifically bindantigen. The term "domain" refers to a globular region of a heavy or light chain polypeptidecomprising peptide loops (e.g., comprising 3 or 4 peptide loops) stabilized, for example, by Bpleated 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 classmembers in the case of "constant" domain, or the significant variation within the domains ofvarious class members in the case of a "variable" domain. Antibody or polypeptide "domains" areoften referred to interchangeably in the antibody or polypeptide "regions". The "constant" domainsof an antibody light chain are referred to interchangeably as "light chain constant regions", "light116chain constant domains", "CL" regions or "CL" domains." The "constant" domains of an antibodyheavy chain are referred to interchangeably as "heavy chain constant regions", "heavy chainconstant domains", "CH" regions or "CH" domains." The "variable" domains of an antibody lightchain are referred to interchangeably as "light chain variable regions", "light chain variabledomains", "VL" regions or "VL" domains." The "variable" domains of an antibody heavy chainare referred to interchangeably as "heavy chain constant regions", "heavy chain constant domains","VH" regions or "VH" domains.

[0249] The term "region" can also refer to a part or portion of an antibody chain or antibodychain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant orvariable domain, as defined herein), as well as more discrete parts or portions of said chains ordomains. For example, light and heavy chains or light and heavy chain variable domains include"complementarity determining regions" or "CDRs" interspersed among "framework regions" or"FRs", as defined herein.

[0250] The term "conformation" refers to the tertiary structure of a protein or polypeptide (e.g.,an antibody, antibody chain, domain or region thereof). For example, the phrase "light (or heavy)chain conformation" refers to the tertiary structure of a light (or heavy) chain variable region, andthe phrase "antibody conformation" or "antibody fragment conformation" refers to the tertiarystructure of an antibody or fragment thereof.

[0251] The term "antibody-like protein scaffolds" or "engineered protein scaffolds" broadlyencompasses proteinaceous non-immunoglobulin specific-binding agents, typically obtained bycombinatorial engineering (such as site-directed random mutagenesis in combination with phagedisplay or other molecular selection techniques). Usually, such scaffolds are derived from robustand small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stablyfolded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin or theankyrin repeat).

[0252] Such scaffolds have been extensively reviewed in Binz et al. (Engineering novelbinding proteins from non-immunoglobulin domains. Nat Biotechnol 2005, 23:1257-1268),Gebauer and Skerra (Engineered protein scaffolds as next-generation antibody therapeutics. CurrOpin Chem Biol. 2009, 13:245-55), Gill and Damle (Biopharmaceutical drug discovery usingnovel protein scaffolds. Curr Opin Biotechnol 2006, 17:653-658), Skerra (Engineered proteinscaffolds for molecular recognition. J Mol Recognit 2000, 13:167-187), and Skerra (Alternative117non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol 2007, 18:295-304), andinclude without limitation affibodies, based on the Z-domain of staphylococcal protein A, a threehelix bundle of 58 residues providing an interface on two of its alpha-helices (Nygren, Alternativebinding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold.FEBS J 2008, 275:2668-2676); engineered Kunitz domains based on small polypeptides of 58residues) and robust, disulphide-crosslinked serine protease inhibitor, typically of human origin(e.g., LACI-D1), which can be engineered for different protease specificities (Nixon and Wood,Engineered protein inhibitors of proteases. Curr Opin Drug Discov Dev 2006, 9:261-268);monobodies or adnectins based on the 10th extracellular domain of human fibronectin III (10Fn3),which adopts an Ig-like beta-sandwich fold (94 residues) with 2-3 exposed loops but lacks thecentral disulphide bridge (Koide and Koide, Monobodies: antibody mimics based on the scaffoldof the fibronectin type III domain. Methods Mol Biol 2007, 352:95-109); anticalins derived fromthe lipocalins, a diverse family of eight-stranded beta-barrel proteins (ca. 180 residues) thatnaturally form binding sites for small ligands by means of four structurally variable loops at theopen end, which are abundant in humans, insects, and many other organisms (Skerra, Alternativebinding proteins: Anticalins-harnessing the structural plasticity of the lipocalin ligand pocket toengineer novel binding activities. FEBS J 2008, 275:2677-2683); DARPins, designed ankyrinrepeat domains (166 residues), which provide a rigid interface arising from typically three repeatedbeta-turns (Stumpp et al., DARPins: a new generation of protein therapeutics. Drug Discov Today2008, 13:695-701); avimers (multimerized LDLR-A module) (Silverman et al., Multivalentavimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol2005, 23:1556-1561); and cysteine-rich knottin peptides (Kolmar" Alternative binding proteins:biological activity and therapeutic potential of cystine-knot miniproteins. FEBS J 2008, 275:2684-2690).

[0253] "Specific binding" of an antibody means that the antibody exhibits appreciable affinityfor a particular antigen or epitope and, generally, does not exhibit significant cross reactivity."Appreciable" binding includes binding with an affinity of at least 25 µM. Antibodies withaffinities greater than 1 x 107 M¹ (or a dissociation coefficient of 1μM or less or a dissociationcoefficient of 1nm or less) typically bind with correspondingly greater specificity. Valuesintermediate of those set forth herein are also intended to be within the scope of the presentdisclosure and antibodies of the disclosure bind with a range of affinities, for example, 100nM or118less, 75nM or less, 50nM or less, 25nM or less, for example 10nM or less, 5nM or less, 1nM orless, or in embodiments 500pM or less, 100pM or less, 50pM or less or 25pM or less. An antibodythat "does not exhibit significant cross reactivity" is one that will not appreciably bind to an entityother than its target (e.g., a different epitope or a different molecule). For example, an antibodythat specifically binds to a target molecule will appreciably bind the target molecule but will notsignificantly react with non-target molecules or peptides. An antibody specific for a particularepitope will, for example, not significantly cross react with remote epitopes on the same proteinor peptide....

Claims

CLAIMSPCT / US2025 / 0122071. An engineered adeno associated virus (AAV) capsid polypeptide comprising a transferrinreceptor (TfR1) binding modification defined by the formula X₁-X2-X3-[7-mer]-X4-Xs-Х6-X7,wherein the 7-mer is one of YSRIGPN (SEQ ID NO: 1390), YSRNSDN (SEQ ID NO: 1391),LHRLGPN (SEQ ID NO: 1392), FRSTNGV (SEQ ID NO: 1393), FVSTNGV (SEQ ID NO:1394), FZ1STNGZ2 (SEQ ID NO: 1395), FRSTNGZ3 (SEQ ID NO: 1396), or VESTNGR (SEQID NO: 1397), and wherein the 7-mer is inserted between amino acids 588 and 589 of an AAV9capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype,and wherein X1, X2, X3, X4, X5, X6, X7 indicate an amino acid modification at one or more aminoacid positions in the capsid polypeptide flanking the inserted 7-mer.

2. The engineered AAV capsid polypeptide of claim 1, wherein the 7-mer comprises orconsists of YSRIGPN (SEQ ID NO: 1390), YSRNSDN (SEQ ID NO: 1391), or LHRLGPN (SEQID NO: 1392), and whereinX1 is A, G, E, L, N, Q, S, W, or M;X2 is A, F, I, L, M, N, Q, P, T, V, or Y;X3 is Q;X4 is any amino acid;Xs is D, F, G, I, L, M, Q, P, S, T, or V;X6 is A, C, F, G, H, I, P, S, T, V, W, or Y;X7 is D, E, Q, or T;or any combination thereof.

3. The engineered AAV capsid polypeptide of claim 2, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 60-1210.

4. The engineered AAV capsid polypeptide of claim 2, wherein the n-mer comprises orconsists of YSRIGPN (SEQ ID NO: 1390), and whereinX₁ is A, G, E, L, N, Q, S, W, or M;X2 is of A, F, I, L, M, N, Q, P, T, V, or Y;2705.X3 is of Q;X4 is of A, E, F, H, I, L, M, N, P, Q, V, Y, D, or G;Xs is of D, F, G, I, L, M, Q, P, S, T, or V;X6 is of A, C, F, G, H, I, P, S, T, V, W, or Y;X7 is of D, E, Q, or T;or any combination thereof.The engineered AAV capsid polypeptide of claim 4, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 60-1136.

6. The engineered AAV capsid polypeptide of claim 2, wherein the n-mer comprises orconsists of YSRNSDN (SEQ ID NO: 1391) andX₁ is of S, or W;X2 is of V, I, or F;X3 is of Q;X4 is of any amino acid;X5 is of Q or T;X6 is of A;X7 is of Q:or any combination thereof.

7. The engineered AAV capsid polypeptide of claim 6, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 1152-1157.

8. The engineered AAV capsid polypeptide of claim 2, wherein the n-mer comprises orconsists of LHRLGPN (SEQ ID NO: 1392) andX₁ is of S, A, L, or М;X2 is of A or P:X3 is of Q;X4 is of A, E, F, H, I, L, M, N, P, Q, V, or Y;Xs is of Q;X6 is of A, P, S, or T;271WO 2025 / 155923X7 is of D, E, Q, or T;or any combination thereof.PCT / US2025 / 0122079. The engineered AAV capsid polypeptide of claim 8, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 1158-1210.

10. The engineered AAV capsid polypeptide of claim 1, wherein the 7-mer comprises orconsists of FRSTNGV (SEQ ID NO: 1393), FVSTNGV (SEQ ID NO: 1394), FZ1STNGZ2 (SEQID NO: 1395), FRSTNGZ3 (SEQ ID NO: 1396), or VESTNGR (SEQ ID NO: 1397) and whereinX₁ is of S:X2 is of A, S, M, or D;X3 is of F, H, I, L, M, N, Q, R, Y, D, or E;X4 is of A, S, or M;X5 is of Q or P;X6 is of A, F, H, Q, or S;X, is of A, D, E, F, Q, S, or T;or any combination thereof.

11. The engineered AAV capsid polypeptide of claim 9, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 1211-1389.

12. The engineered AAV capsid polypeptide of claim 9, wherein the n-mer comprisesconsists of FRSTNGV (SEQ ID NO: 1393), andX₁ is of S;X2 is of A or S;X3 is of D;X4 is of A, or S;X5 is of Q or P;X6 is of A, F, H, Q, or S;X7 is of D, E, Q, or T;or any combination thereof.272or13 The engineered AAV capsid polypeptide of claim 12, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 1211-1266.

14. The engineered AAV capsid polypeptide of claim 9, wherein the n-mer comprises orconsists of FVSTNGV (SEQ ID NO: 1394), andX₁ is of S;X2 is of A, S, or M;X3 is of Q, E, or D;X4 is of A, or M;X5 is of Q or P;X6 is of А;X7 is of E:or any combination thereof.

15. The engineered AAV capsid polypeptide of claim 14, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 1267-1298.

16. The engineered AAV capsid polypeptide of claim 9, wherein the n-mer comprises orconsists of FZ1STNGZ2 (SEQ ID NO: 1395) or FRSTNGZ3 (SEQ ID NO: 1396), andX₁ is of S;X2 is of A or S:X3 is of Q or D;X4 is of A;Xs is of Q;X6 is of А;X7 is of E;or any combination thereof.

17. The engineered AAV capsid polypeptide of claim 16, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 1299-1319.27318 The engineered AAV capsid polypeptide of claim 9, wherein the 7-mer comprises orconsists of VESTNGR (SEQ ID NO: 1397), andX₁ is of S;X₂ is of S or D;X3 is of F, H, I, L, M, N, Q, R, or Y;X4 is of A;Xs is of Q or P;X6 is of A;X7 is of A, D, E, F, Q, S, or T;or any combination thereof.19 The engineered AAV capsid polypeptide of claim 17, wherein the TfR1 bindingmodification comprises or consists of the amino acid sequence of one of SEQ ID Nos: 1320-1388.

20. The engineered AAV capsid polypeptide of any one or the preceding claims, whereinZi is selected from the group consisting of A, D, H, N, Q, and S; Z2 is selected from the groupconsisting of K and R; and Z3 is selected from the group consisting of L, M, and R.21 The engineered AAV capsid polypeptide of claim 1, wherein the TfR1 bindingmodification comprises or consists of an amino acid sequence of EAQYSRIGPNNQAQ:SADFRSTNGVAQAE; or GAQYSRIGPNPQPE.22 The engineered AAV capsid polypeptide of any of the preceding claims, wherein theviral capsid protein is VP1, VP2, VP3, or a combination thereof.

23. The engineered AAV capsid polypeptide of any of the preceding claims, wherein theother AAV serotype is AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8,AAV rh.74, or AAV rh. 10.27424. An engineered AAV particle comprising the engineered AAV capsid polypeptide ofany one of the preceding claims, and further comprising a recombinant AAV genome encoding atransgene.

25. The engineered AAV particle of claim 24, wherein the transgene encodes a therapeuticpolypeptide, an antibody or fragment thereof, a siRNA, a CRISPR-Cas system, TranscriptionActivator-like Effector (TALE)- or Zinc Finger Protein (ZFP)-based transcriptional activator;repressor; or epigenomic silencer, a RNA encoding a partial gene fragment designed fortransplacing into an endogenous RNA, one or more transfer RNAs, or a component thereof, or anOMEGA system or any component thereof.

26. The engineered AAV particle of claims 24 or 25, wherein the transgene is operablylinked to a regulatory sequence that promotes expression in the CNS.

27. A pharmaceutical composition comprising the recombinant engineered AAV particleof any one of claims 24 to 26 and an acceptable carrier.

28. A method of delivering a polypeptide or polynucleotide to the CNS of a subjectcomprising administering the pharmaceutical composition of claim 27 to the subject.

29. The method of claim 28, wherein the pharmaceutical composition is delivered at adosage between 0.1 x 1012 vg / kg to 1 x 1014 vg / kg.

30. The method of claim 29, wherein the dosage is between 0.1 x 1012 vg / kg to 100 x 1012vg / kg.

31. The method of claim 30, wherein the dosage is between 1 x 1012 vg / kg to 10 x 1012vg / kg.

32. The method of claim 31, wherein the dosage is 5 x 1012 vg / kg.

33. The method of claim 31, wherein the dosage is 2 x 1012 vg / kg.27534. The method of any one of claims 28 to 33, wherein the pharmaceutical composition isadministered systemically or directly to the CNS.

35. A method of manufacturing a recombinant engineered AAV, said method comprising:culturing mammalian cells comprising; (1) a polynucleotide encoding the engineeredAAV capsid polypeptide of any one of claims 1 to 26, (2) a polynucleotide encoding a recombinantAAV genome comprising a transgene operably linked to a regulatory sequence and flanked byAAV ITR sequences, and optionally (3) polynucleotide encoding adenoviral helper genes, underconditions sufficient for production of recombinant engineered AAV particles; andrecovering the recombinant engineered AAV particles from said culture.

36. A host cell for production of recombinant engineered AAV particles which comprisesa polynucleotide encoding the engineered AAV capsid polypeptide of any one of claims 1 to 26, apolynucleotide encoding a recombinant AAV genome comprising a transgene operably linked toregulatory sequence and flanked by AAV ITR sequences, and optionally a polynucleotideencoding adenoviral helper genes.a37. An AAV library comprising a population of variant engineered recombinant AAVparticles wherein each member of the population of variant engineered recombinant AAV particlescomprises a variant recombinant AAV capsid polypeptide having a modification defined by theformula X₁-X2-X3-[7-mer]-X4-X5-X6-X7, and wherein the 7-mer is inserted between amino acids588 and 589 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptideof another AAV serotype, and wherein X1, X2, X3, X4, X5, X6, X7 indicate modifications at one ormore amino acid positions in the capsid polypeptide flanking the inserted 7-mer, wherein themodification has been selected for binding of TfR1 and / or increased tropism for the CNS relevantto a reference AAV particle without the modification.

38. The AAV library of claim 37, wherein the AAV serotype is AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10.

39. A method of screening an AAV library for a recombinant AAV particle which bindsTfr1 and / or has increased tropism for the CNS, said method comprising assaying the AAV library276of claim 37 for increased binding of Tfr1 and / or tropism for the CNS relative to an AAV vectorwith a reference capsid and selecting those recombinant AAV vectors which have increasedbinding of Tfr1 and / or tropism for the CNS.

40. A method for training a machine learning algorithm comprising:a. receiving, by at least one computing device, a plurality of AAV capsid polypeptidesequences comprising a modification for binding to a TfR1;b. training, by at least one computing device, with the plurality of AAV capsid polypeptidesequences comprising a modification for binding to a TfR1, a transferrin receptor (TfR1)targeting machine learning model; andC. deploying, by at least one computing device, the TfR1 targeting machine learningalgorithm.41 The method of claim 40, wherein the TfR1 targeting machine learning model is trainedto identify one or more sequences from the plurality of sequences within increased binding of Tfr1.

42. The method of any one of claims 40-41, wherein the TfR1 targeting machine learningmodel is trained to identify one or more sequences from the plurality of sequences that decreasestransduction of off-target tissues.

43. The method of any one of claims 40-42, wherein the TfR1 targeting machine learningmodel is trained to identify one or more sequences from the plurality of sequences that binds to aextracellular domain of TFRC.

44. The method of any one of claims 40-43, wherein the TfR1 targeting machine learningmodel is trained to identify one or more sequences from the plurality of sequences that binds to aapical domain.

45. The method of any one of claims 40-44, wherein the modification is defined by theformula X₁-X2-X3-[7-mer]-X4-X5-X6-Х7.27746. The method of any of claims 40-45, wherein the training comprises unsupervisedlearning, supervised learning, semi-supervised learning, reinforcement learning, transfer learning,incremental learning, curriculum learning, learning to learn, or contrastive learning.

47. The method of claim 40-46, wherein the transferrin receptor (TfR1) targeting machinelearning model comprises of linear classifiers, logistic classifiers, random forest, artificial neuralnetworks, matrix factorization, support vector machines, K-means clustering, or K-nearestneighbor.

48. The method of claim 40-46 wherein the transferrin receptor (TfR1) targeting machinelearning model comprises of Boltzmann machines, Bayesian networks, autoregressive models,variational auto encoders (VAEs), diffusion models, energy-based models, flow-based models,generative adversarial networks (GANs), mixture models, hidden Markov models, or largelanguage models (LLMs).

49. The method of claim 40-46 wherein the transferrin receptor (TfR1) targeting machinelearning model comprises of convolutional neural networks (CNNs), recurrent neural networks(RNNs), long short-term memory models (LSTMs), gated recurrent units (GRUs), capsulenetworks, attention mechanisms, or transformer networks.

50. The method of claim 40-47, the transferrin receptor (TfR1) targeting machine learningmodel is a pre-trained, and further trained to predict transferrin receptor (TfR1) targeting by aplurality of sequences.

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