Methods and systems directed to therapeutic delivery of the human GBA1 gene

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-hTFR1, that binds human Transferrin Receptor (TfR1), a transporter expressed on the blood-brain barrier (BBB). BI-hTFR1 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-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9. Our work establishes BI-hTFR1 as a promising vector for human CNS gene therapy.

Description

METHODS AND SYSTEMS DIRECTED TO THERAPEUTIC DELIVERY OF THEHUMAN GBA1 GENECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 612,232, filed December 19, 2023. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Nos. NS111689 and MH120096 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] Reference is made to the electronic sequence listing ("BROD-5990WP_ST26.xml"; Size is 28,896,584 bytes, created on December 11, 2024) is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to treating lysosomal storage disease orders by delivering GBAI to the central nervous system (CNS).BACKGROUND

[0005] The development of gene therapies for neurodevelopmental and neurological disorders has been constrained by the inability to efficiently deliver genes throughout the CNS. Several studies have reported engineered AAV9 capsids, most notably the AAV-PHP.B family, that are capable of highly effective gene transfer throughout the CNS after intravenous administration in adult mice. To date however, none of the engineered AAV capsids that cross the blood-brain barrier (BBB) and transduce the mouse brain with high efficiency have been shown to exhibit their enhanced CNS tropism in primates. In this work, Applicants take a mechanism-first approach and engineer AAV capsids that interact with the Transferrin Receptor (TFRC).

[0006] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0007] In some aspects, the techniques described herein relate to an engineered recombinant adeno associated virus (AAV) particle including an engineered AAV capsid polypeptide including a transferrin receptor (TfR1) binding moiety and a recombinant AAV genome including a polynucleotide encoding GBA1 operably linked to a regulatory element that promotes expression of GBA1 in the central nervous system (CNS) of said subject flanked by AAV ITR sequences.

[0008] In some aspects, the techniques described herein relate to an engineered recombinant AAV particle, wherein the TfR1 binding moiety is inserted or substituted in loop IV, loop VIII, or both an AAV capsid protein.

[0009] In some aspects, the techniques described herein relate to an engineered recombinant AAV particle, wherein the AAV capsid polypeptide is VP1, VP2, VP3, or a combination thereof.

[0010] In some aspects, the techniques described herein relate to an engineered recombinant AAV particle, wherein the TfR1 binding moiety is inserted between amino acids 588 and 589 of a capsid protein of AAV9, or in an analogous position of a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10.

[0011] In some aspects, the techniques described herein relate to an engineered recombinant AAV particle, wherein the TfR1 binding moiety is inserted between two consecutive amino acids within amino acid 451-460 of a capsid protein of AAV9, or in an analogous position of a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10.

[0012] In some aspects, the techniques described herein relate to an engineered recombinant AAV particle, wherein the TfR1 binding moiety includes of a 7-mer, the 7-mer includes of LHRLGPN (SEQ ID NO: 36834), YSRIGPN (SEQ ID NO: 42487), LHRLGPD (SEQ ID NO: 36413), LHRAGPD (SEQ ID NO: 36894), YSRIGPD (SEQ ID NO: 38223), LSRIGPD (SEQ ID NO: 36274), LARSGPD (SEQ ID NO: 18035), YSRNSDN (SEQ ID NO: 42488), LHKAGPN (SEQ ID NO: 36305), LSRIGPN (SEQ ID NO: 36347), LAKSGPN (SEQ ID NO: 36287), YARNGPN (SEQ ID NO: 14048), FRSTNGV (SEQ ID NO: 16070), VESTNGR (SEQ ID NO: 36431), VDSTNGV (SEQ ID NO: 12206), VQSTNGV (SEQ ID NO: 36423), VSSTNGV (SEQ ID NO: 12333), TESTNGR (SEQ ID NO: 17558), VQSTNGI (SEQ ID NO: 11292), FVSTNGV (SEQ ID NO: 42491), RGEDVYP (SEQ ID NO: 36864), RLEDVFP (SEQ ID NO: 36264), RTYDSYP (SEQ ID NO: 37938), RTYDAYP (SEQ ID NO: 38571), RTYDSFP (SEQ ID NO: 37806), RTETVYP (SEQ ID NO: 36486), RTETVFP (SEQ ID NO: 36389), RTEHVFP (SEQ ID NO: 36603), LCKPCLD (SEQ ID NO: 36437), LCKPCPT (SEQ ID NO: 36438), KDEFTTF (SEQ ID NO: 36308), KDDFTTY (SEQ ID NO: 36336), RDEFTTY (SEQ ID NO: 36615), KDEFSTY (SEQ ID NO: 36390), RDEFTSF (SEQ ID NO: 36701), REDHVSW (SEQ ID NO: 37067), IALKGWD (SEQ ID NO: 36248), NALEGRD (SEQ ID NO: 36407), VALEGRD (SEQ ID NO: 36604), VALKGWD (SEQ ID NO: 17701), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), YHRLSNN (SEQ ID NO: 16636), VHRLQDK (SEQ ID NO: 16602) LHALSHN (SEQ ID NO: 16608), PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16199), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 16206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGI (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSNGV (SEQ ID NO: 16123).

[0013] In some aspects, the techniques described herein relate to an engineered recombinant AAV particle, wherein the 7-mer includes YSRIGPN (SEQ ID NO: 42487).

[0014] In some aspects, the techniques described herein relate to an engineered recombinant AAV particle, wherein the Tfr1 binding moiety is selected from any of SEQ ID NO: 42513-43842.

[0015] In some aspects, the techniques described herein relate to a pharmaceutical composition including the engineered recombinant AAV particle of any described herein and an acceptable carrier.

[0016] A method of delivering GBAl to the central nervous system (CNS) comprising administering the pharmaceutical composition of any of those described herein.

[0017] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition is administered at a dosage of between 0.1 x 1012 vg / kg to 1 x 1014 vg / kg.

[0018] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition is administered at a dosage of 0.1 x 1012 vg / kg to 100 x 1012 vg / kg.

[0019] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition is administered at a dosage of 1 x 1012 vg / kg to 10 x 1012 vg / kg.

[0020] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition is administered at a dosage of 5 x 1012 vg / kg.

[0021] In some aspects, the techniques described herein relate to a method, wherein the subject suffers from Gaucher's disease.

[0022] In some aspects, the techniques described herein relate to a method, wherein the subject suffers from Parkinson's disease.

[0023] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0025] 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 CAG-NLS-mScarlet-P2A- Luciferase-WPRE-SV40pA construct 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 determine significant differences in transduction among five AAV variants on five CHO cell lines with AAV9 as the main comparison group for each cell line and using a Bonferroni multiple comparison correction: **** and *** indicate p < 0.0001 and ≤ 0.001, respectively; n = 3 replicates, error bars indicate° SEM. The TfR1-binding variants exhibited enhanced (1D) association with and (1E) transduction (Luciferase activity) of human brain microvascular endothelial cells (hBMVEC) and hCMEC / D3 cells. Values are normalized to AAV9 in each cell line. One-way ANOVA test to determine significant differences among five AAV variants with AAV9 as the comparison group for each cell line and using a Dunnett's multiple comparison correction: ****, ***, and * indicate p ≤ 0.0001, ≤ 0.001, and ≤ 0.05, respectively; n = 4 replicates, error bars indicate + SEM. (1F) The binding kinetics between each capsid and full-length human TfR1 were assessed by BLI. AAVx probes were loaded with capsid and human TfR1 was used as an analyte. Sensorgram curve fits (black dashdot lines) were generated by applying global 2:1 exponential association and decay models.

[0026] FIG. 2A-2D – Human TfR1-targeted capsids transduce human brain endothelial cells via interactions with the apical domain of human TfR1. (2A) The plots show transduction (Luciferase, normalized RLU) of hCMEC / D3 cells incubated with 3 x 108 vg / mL of the indicated AAV and the specified concentrations of the OKT9 or AF2474 antibody. Two-way ANOVA test to determine significant differences in transduction with AAV9 or BI-hTFR1 and one of two antibodies at different concentrations with the no antibody control for each condition as the main comparison 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 to full-length human TfR1 was assessed by BLI. AAVx probes were loaded with AAV9 or BI- hTFR1. Human TfR1 that either had or had not been pre-incubated with 300 nM holo-Tf was used as an analyte. 2:1 binding model curve fits (black dash-dot lines) are shown. (2C) Biotinylated holo-Tf was immobilized on streptavidin-coated BLI probes (SA probe), introduced first into human TfR1, and then into buffer with or without the OKT9 antibody, and finally into BI-hTFR1 virus particles. Segments shaded in gray highlight the indicated association step. (2D) BI-hTFR1 or AAV2 was incubated with hCMEC / D3 cells at 50,000 vg / cell for one hour at 4°C, with or without OKT9 (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).

[0027] FIG. 3A-3C – BI-hTFR1 is efficiently endocytosed and actively transported across human brain endothelial cells. (3A) Schematic shows the pooled transwell BBB model experimental 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) BI- hTFR1 or AAV2 were incubated with hCMEC / D3 cells at 25,000 vg / cell for one hour at 37°C and stained for endosomal markers Rab5 and Rab7 as well as the AAVs. Scale bar = 15 µm.

[0028] 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 have been replaced by those of human TFRC. (4B) BI-hTFR1 or AAV9 encoding CAG-NLS-mScarlet- P2A-Luciferase-WPRE-SV40pA were intravenously injected into adult female C57BL / 6J_or TFRC 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) Luciferase activity within different organs are shown at three weeks post-injection. Two-way ANOVA test to determine significant differences within organs among the four groups of mice with BI-hTFR1 in TFRC KI mice as the main comparison group and using a Bonferroni multiple comparison 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.

[0029] FIG. 5A-5F – BI-hTFR1 efficiently transduces neurons and astrocytes throughout the CNS. Representative (5A) whole brain and (5B) spinal cord images from each group of mice at three weeks post-injection are shown. Representative images show cells transduced by BI- hTFR1 overlaid with (5C) NeuN+ or (5E) SOX9+ stained cells in the cortex, thalamus, and striatum of TFRC KI mice. The percentages of (5D) NeuN⁺ neurons or (5F) SOX9+ astrocytes that expressed mScarlet in the cortex, striatum, and thalamus are shown. Two-way ANOVA test to determine significant differences in transduction within specific brain regions among the four groups of mice with BI-hTFR1 in TFRC KI mice as the main comparison group and using a Bonferroni multiple comparison correction: **** indicates p ≤ 0.0001; each data point represents an individual mouse, error bars indicate ± SEM.

[0030] FIG. 6A-6D – BI-hTFR1 efficiently delivered GBAI and increased GCase activity in the brains of TFRC KI mice. (6A) Schematic of the experiment shows the ssDNA AAV genome expressing human glucocerebrosidase that was packaged into AAV9 or BI-hTFR1 and administered to TFRC KI transgenic mice at either 1 x 1014 vg / kg or 5 x 1012 vg / kg. (6B) The biodistribution of AAV genomes found in brain and liver tissue relative to AAV9 (n = 4 mice per condition) is shown. One-way ANOVA with Sidak's multiple comparison post-hoc test using AAV9 as the control group: **** and *** indicate p < 0.0001 and ≤ 0.001, respectively; each data point represents an individual mouse, error bars indicate ± SEM. (6C) Sagittal brain sections (top) from the mice in each group show GBA-HA in whole brain sagittal sections. Scale bar = 1 mm. Images of immunostaining (bottom) show neurons (NeuN) and GBA-HA in the substantia nigra pars compacta. Scale bar = 25 µm. (6D) GCase enzyme activity levels in brain and liver tissue homogenate, CSF, and serum are shown. One-way ANOVA with Sidak's multiple comparison post-hoc test with AAV9 as the control group: **** indicates p < 0.0001; each data point represents an individual mouse, error bars indicate ± SEM.

[0031] FIG. 7A-7B – Validation assays confirm expression of TfR1 from the indicated species in stable CHO cell lines. (7A) Species-specific primers (table S1) were used to assess TFRC or Tfrc mRNA expression levels by RT-qPCR. Each species primer set is shown with the indicated color (legend, top). The graph shows qPCR cycle threshold (Ct) values showing selective early cycle number amplification of the target species sequence. The bar indicates the mean Ct value (n = 3 replicates per cell line). (7B) Images show immunofluorescence for TfR1. Each cell line was fixed, permeabilized, and stained with the indicated anti-TfR1 antibodies to assess the expression of exogenous TfR1.

[0032] FIG. 8A-8D – The binding between BI-hTFR1 and human TfR1 is inhibited by the apical domain binding antibody OKT9 but not by holo-Tf. (8A) To assess binding between each AAV capsid and full-length human TfR1, AAVx probes were loaded with capsid and TfR1 was used as an analyte. Average kinetic constants and standard errors were computed from triplicate 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 and dissociation in the presence of the indicated concentration of OKT9. The gray curve shows OKT9 binding to Fc-hTfR1 in the absence of BI-hTFR1. (8C) The binding kinetics between full-length human TfR1 and human holo-Tf were assessed by BLI. Mouse Fc (MFC) probes were loaded with an anti-transferrin antibody, 12A6. Holo-Tf was loaded onto the probe and associated with serial dilutions of TfR1 analyte. (8D) The effect of Tf on BI-hTFR1 binding to full-length human TfR1 was assessed by BLI. AAVx probes were loaded with AAV9 or BI-hTFR1. Human TfR1 that either had or had not been pre-incubated with 300 nM holo-Tf was used as an analyte. Average kinetic constants and standard errors were computed from triplicate trials.

[0033] 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. Cells were then fixed, permeabilized, and stained for AAV and nuclei. AAV2 was chosen as a control for subsequent experiments because AAV9 immunostaining was nearly undetectable. (9B) BI-hTFR1 or AAV2 was incubated with hCMEC / D3 cells at 30,000 vg / cell for one hour at 37°C. Cells were fixed, permeabilized, and stained for AAV and TfR1. (9C) BI-hTFR1 or AAV2 was incubated with hCMEC / D3 cells at 25,000 vg / cell in prechilled media containing Tf-647 (1 µg / mL) for one hour at 4°C. Cells were then fixed and stained for AAV, Tf, and nuclei. All scale bars = 15 µm.

[0034] FIG. 10 – AAV colocalization with subcellular compartment markers. BI-hTFR1 or AAV2 was incubated with hCMEC / D3 cells at 25,000 vg / cell for one hour at 37°C. Cells were then fixed, permeabilized, and stained for AAV, endosomes (Rab5 and Rab7), cis-Golgi (RCAS1), trans-Golgi network (TGN46) or endoplasmic reticulum (KDEL), and nuclei. All scale bars = 15 μm.v

[0035] FIG. 11A-11B – TfR1 levels in TFRC KI mice match those in WT mice. (11A) RT- qPCR using primers spanning exon 1 and exon 2 of the mouse Tfrc gene (table S1) was performed to assess mRNA levels in TFRC KI mice or C57BL / 6J controls treated with either AAV9 or BI- hTFR1 (n = 3 or 4 animals per group; error bars indicate ± SEM). No significant difference in TfR1 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 TFRC KI homozygous mice injected with AAV9 or BI-hTFR1 and purified full-length human or mouse TfR1 Fc-fusion proteins with N-terminal flag tag were stained using a polyclonal anti-human TfR1 antibody, AF2474 (R&D systems), or a monoclonal antibody, H68.4 (Thermofisher, Catalog #13- 6890) that recognizes mouse and human TfR1.

[0036] FIG. 12A-12C – BI-hTFR1 exhibited a CNS-specific enhanced tropism in TFRC KI mice and similar transduction to AAV9 in the liver and dorsal root ganglion. BI-hTFR1 or AAV9:CAG-NLS-mScarlet-P2A-Luciferase-WPRE-SV40pA was intravenously injected into adult female C57BL / 6J or TFRC KI mice at a dose of 5 x 1011 vg / mouse. The (12A) mScarlet transcript levels relative to AAV9 in C57BL / 6J mice, and native fluorescence in the (12B) liver and (12C) dorsal root ganglion at three weeks post-injection are shown. For (12A), two-way ANOVA to determine significant differences within organs among the four groups of mice with BI-hTFR1 in TFRC KI mice as the main comparison group and using a Bonferroni multiple comparison 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.

[0037] FIG. 13A-13C – Expression of GBA-HA in the liver and brain regions of mice injected with AAV9 or BI-hTFR1:GBAI. (13A) Images show NeuN and HA immunostaining in the deep cerebellar nuclei and brainstem in TFRC KI mice with the indicated treatment conditions. Scale bar = 25 µm. (13B) The cell counts of transduced NeuN+ cells expressing GBA- HA in the cortex, striatum, and thalamus of TFRC KI mice injected with 1 x 1014 vg / 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 the indicated conditions are shown. Scale bar = 50 µm.

[0038] FIG. 14 – AAV9 capsid residues 539-605 aligned to other previously described capsids. (SEQ ID NO: 43879-43898) The 7-mer insertion site between AAV9 residue 588 and 589 is shown. The black bars above the alignment highlight surrounding residues that were modified in this study. Corresponding residues in other example capsid sequences are outlined and residues that differ from AAV9 are shown in gray. Sequences were aligned using MUSCLE (SnapGene).

[0039] FIG. 15 – TfR1 binding modification (SEQ ID NO: 43899-43911) – example insertions between residues 558 and 559 AAV9 VP1.

[0040] FIG. 16A-16E – Partial views of example serotype sequence alignment (SEQ ID NO: 43912-43924).

[0041] FIG. 17 – TfR1 binding moiety insertion site in AAV9 VP1 capsid (SEQ ID NO: 43925-43928).

[0042] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0043] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (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 by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0044] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0045] The term "optional" or "optionally" means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0046] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0047] The terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / - 1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0048] As used herein, a "biological sample" may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a "bodily fluid". The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0049] The terms "subject," "individual,” and “patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0050] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to "one embodiment”, “an embodiment," "an example embodiment," means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," or "an example embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0051] The term "polypeptide" and "protein" are used interchangeably herein to refer to a viral capsid unless indicated otherwise.

[0052] Reference is made to PCT / US2023 / 070285, hereby incorporated by reference.

[0053] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0054] In some aspects, the techniques described herein relate to an engineered recombinant adeno associated virus (AAV) particle comprising an engineered AAV capsid polypeptide comprising a transferrin receptor (TfR1) binding moiety and a recombinant AAV genome comprising a polynucleotide encoding GBA1 operably linked to a regulatory element that promotes expression of GBA1 in the central nervous system (CNS).

[0055] The TFRC (i.e., TfR1 protein encoded by the TFRC gene, or CD71) comprises of two types of receptors: TFRC1 (or cluster of differentiation 71 (CD71)) and TFRC2. TFRC1 binds transferrin (TF) with high affinity and is commonly expressed. TFRC1 is a type II transmembrane glycoprotein of around 90 kDa and comprises of around 760 amino acids. TFRC1 is typically found as a dimer linked by disulfide bonds on the cell surface, see FIG. 4. The TFRC1 domain contains an extracellular C-terminal domain (around 671 amino acids) and comprises of the TF binding site. The extracellular C-terminal domain comprises of three subdomains: apical, helical, and protease-like domain, see FIG. 4. Furthermore, the extracellular C-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 adequate function of the receptor. TFRC1 additionally comprises of a transmembrane domain (around 29 amino acids), and an intracellular N-terminal domain (around 61 amino acids). Alternative to the delivery of iron through TF with TFRC1, uptake of iron can also occur through H-ferritin binding through the apical domain. See also Candelaria, P. V.; et al. Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-Cancer Agents. Frontiers in Immunology, 2021, 12, hereby incorporated by reference.

[0056] In an example embodiment, the TfR1 binding moiety binds to the extracellular domain of TFRC. In an example embodiment, the TfR1 binding moiety binds to one or more of the apical, helical, and / or protease-like domain. In an example embodiment, the TfR1 binding moiety binds to the apical domain.Engineered Viral Particles For Delivery of GBA1

[0057] Described herein are various embodiments of engineered recombinant viral particles, such as adeno-associated virus (AAV) particles, that can be engineered to confer cell-selective tropism, such as CNS tissue- and cell-specific tropism, to an engineered viral particle. The engineered recombinant AAV (rAAV) may comprise a capsid polypeptide engineered to comprise a targeting moiety that is displayed on an outer surface of an assembled rAAV capsid and confers cell-specific tropism, for example, CNS tissue specific tropism and a recombinant AAV genome comprising a polynucleotide encoding GBA1. In an embodiment, the targeting moiety binds human transferrin receptor (TfR1), a transporter expressed on the blood-brain barrier (BBB).Engineered Viral Capsid Polypeptides

[0058] The TfR1 binding moiety is part of a viral capsid protein. The engineered viral capsids can be variants of wild-type viral capsids. In an embodiment, the engineered viral capsid polypeptide is an engineered AAV capsid polypeptide. For example, the engineered AAV capsids can be variants of wild-type AAV capsids. In an embodiment, the wild-type AAV capsids can be composed of VP1, VP2, and VP3 capsid proteins or a combination thereof. In other words, the engineered AAV capsids can include one or more variants of wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. In an embodiment, the serotype of the reference wild-type AAV capsid can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In an embodiment, the serotype of the wild-type AAV capsid can be AAV- 9. The engineered AAV capsids can have a different tropism than the reference wild-type AAV capsid.

[0059] The TfR1 binding moiety may be inserted between two amino acids of the viral polypeptide such that the TfR1 binding moiety is external to (i.e., is presented on the surface of) an assembled rAAV particle. In an example embodiment, the TfR1 binding moiety disclosed herein can be inserted between two amino acids in the wild-type viral protein (VP) (or capsid protein), including in regions that are surface exposed when incorporated into a viral capsid.

[0060] In an embodiment, the TfR1 binding moiety can be inserted between two amino acids in an AAV capsid polypeptide's variable amino acid region. The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (betaB to betal) and an alpha-helix (alphaA) that are conserved in autonomous parvovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structural variable regions (VRs), also called "loops," occur in the surface loops that connect the beta-strands, which cluster to produce local variations in the capsid surface. AAVs have 12 variable regions (also referred to as hypervariable regions) (see, e.g., Weitzman and Linden. 2011. “Adeno-Associated Virus Biology." In Snyder, R.O., Moullier, P. (eds.) Totowa, NJ: Humana Press). In an embodiment, one or more TfR1 binding moieties can be inserted between two amino acids in one or more of the 12 variable regions in the wild-type AVV capsid proteins. In an embodiment, one or more TfR1 binding moieties can each be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X, VR- XI, VR-XII, or a combination thereof. In an example embodiment, the TfR1 binding moiety is inserted or substituted in loops IV and / or VIII.

[0061] In an embodiment, the TfR1 binding moiety is inserted between amino acids 588 and 589 of a capsid protein of AAV9 or in an analogous position of a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10. In one embodiment, the engineered capsid is a modified AAV1 capsid. It can have a TfR1 binding moiety motif inserted after or a neighbor of amino acid 590 (i.e., between amino acids 590 and 591). In an embodiment, the engineered capsid is a modified AAV3 capsid and can have a TfR1 binding moiety motif inserted after or a neighbor of amino acid 586. In an embodiment, the engineered capsid is a modified AAV4 capsid and can have a TfR1 binding moiety motif inserted after or a neighbor of amino acid 586. In an embodiment, the engineered capsid is a modified AAV5 capsid and can have a TfR1 binding moiety motif inserted after or a neighbor of amino acid 575. In an embodiment, the engineered capsid is a modified AAV6 capsid and can have a TfR1 binding moiety inserted at or a neighbor of amino acid 585 and optionally Y705-731, T492V, K531E. In an embodiment, the engineered capsid is a modified AAV8 capsid and can have a TfR1 binding moiety inserted after or a neighbor of amino acids 585 and 590. In an embodiment, the engineered capsid is a modified AAV9 capsid and can have a TfR1 binding moiety inserted in between amino acids 588 and 589. (Büning, H.; Srivastava, A. Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors. Molecular Therapy - Methods & Clinical Development 2019, 12, 248-265). In an embodiment, the engineered capsid can have a TfR1 binding moiety inserted between amino acids 588 and 589 of an AAV9 viral protein. SEQ ID NO: 20506 is a reference AAV9 capsid sequence referencing at least the insertion sites discussed above. In an embodiment, the engineered capsid can have a TfR1 binding moiety inserted between two consecutive amino acids within amino acids 451-460 of a capsid protein of AAV9 viral protein. It will be appreciated that TfR1 binding moieties can be inserted in analogous positions in AAV viral proteins of other serotypes, such as but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, AAV rh. 10 capsid polypeptides.

[0062] In one embodiment, the first 1, 2, 3, or 4 amino acids of a TfR1 binding moiety can replace 1, 2, 3, or 4 amino acids of a polypeptide into which it is inserted and preceding the insertion site. Using an AAV as another non-limiting example, one or more of the TfR1 binding moieties can be inserted into, e.g., an AAV9 capsid polypeptide between amino acids 588 and 589, and the insert can replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the TfR1 binding moiety after insertion is residue 585. It will be appreciated that this principle can apply in any other insertion context and is not necessarily limited to insertion between residues 588 and 589 of an AAV9 capsid or equivalent position in another AAV capsid. It will further be appreciated that in an embodiment, no amino acids in the polypeptide into which the TfR1 binding moiety is inserted are replaced by the TfR1 binding moiety. In an example embodiment, the AAV capsid protein is selected from SEQ ID NO: 20506.

[0063] In an example embodiment, the TfR1 binding moiety is inserted or substituted in loop IV, loop VIII, or both of an AAV capsid protein.

[0064] In an example embodiment, the TfR1 binding moiety includes a 7-mer. In an example embodiment, the 7-mer comprises LHRLGPN (SEQ ID NO: 36834), YSRIGPN (SEQ ID NO: 42487), LHRLGPD (SEQ ID NO: 36413), LHRAGPD (SEQ ID NO: 36894), YSRIGPD (SEQ ID NO: 38223), LSRIGPD (SEQ ID NO: 36274), LARSGPD (SEQ ID NO: 18035), YSRNSDN (SEQ ID NO: 42488), LHKAGPN (SEQ ID NO: 36305), LSRIGPN (SEQ ID NO: 36347), LAKSGPN (SEQ ID NO: 36287), YARNGPN (SEQ ID NO: 14048), FRSTNGV (SEQ ID NO: 16070), VESTNGR (SEQ ID NO: 36431), VDSTNGV (SEQ ID NO: 12206), VQSTNGV (SEQ ID NO: 36423), VSSTNGV (SEQ ID NO: 12333), TESTNGR (SEQ ID NO: 17558), VQSTNGI (SEQ ID NO: 11292), FVSTNGV (SEQ ID NO: 42491), RGEDVYP (SEQ ID NO: 36864), RLEDVFP (SEQ ID NO: 36264), RTYDSYP (SEQ ID NO: 37938), RTYDAYP (SEQ ID NO: 38571), RTYDSFP (SEQ ID NO: 37806), RTETVYP (SEQ ID NO: 36486), RTETVFP (SEQ ID NO: 36389), RTEHVFP (SEQ ID NO: 36603), LCKPCLD (SEQ ID NO: 36437), LCKPCPT (SEQ ID NO: 36438), KDEFTTF (SEQ ID NO: 36308), KDDFTTY (SEQ ID NO: 36336), RDEFTTY (SEQ ID NO: 36615), KDEFSTY (SEQ ID NO: 36390), RDEFTSF (SEQ ID NO: 36701), REDHVSW (SEQ ID NO: 37067), IALKGWD (SEQ ID NO: 36248), NALEGRD (SEQ ID NO: 36407), VALEGRD (SEQ ID NO: 36604), VALKGWD (SEQ ID NO: 17701), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), YHRLSNN (SEQ ID NO: 16636), VHRLQDK (SEQ ID NO: 16602) LHALSHN (SEQ ID NO: 16608), PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16199), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 16206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGI (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSNGV (SEQ ID NO: 16123). In one embodiment, the 7-mer comprises YSRIGPN (SEQ ID NO: 42487).

[0065] In an embodiment, the target binding moiety may be defined by the formula X1-X2-X3- [7-mer]-X4-X5-X6-X7 (Formula I), wherein the 7-mer is inserted between amino acids 588 and 589 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein X1, X2, X3, X4, X5, X6, X7 indicate one or more modifications at amino acid positions in the capsid polypeptide flanking the inserted 7-mer. In an embodiment, the TfR1 binding moiety according to Formula I is selected from any of SEQ ID NO: 42513-43842.Further Capsid Modifications

[0066] In an embodiment, the viral capsid protein may comprise one or more mutations relative to wild type. In an example embodiment, the one or more mutations comprise a K449R substitution in a capsid polypeptide of AAV 9 20507 or a substitution in an analogous position of a capsid polypeptide from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10. In an example embodiment, the K449R substituted AAV capsid is selected from SEQ ID NO: 20507.

[0067] In example embodiments, the viral capsid protein may comprise additional targeting motifs in addition to the n-mer motifs of the present disclosure. Without being bound by theory, the additional TfR1 binding moieties can be antibodies or fragments thereof. In an embodiment, the additional TfR1 binding moiety can be any molecule or composition capable of recognizing, binding, attaching to, or otherwise interacting with a binding partner that can be present on the surface of a target cell. Binding partners include but are not limited to nucleic acids, proteins, peptides, sugars, fats, or any combination thereof or any other molecule or molecules present on a target cell's surface. In an embodiment, the binding partner is unique to a cell type, cell state, or group of related cell types or states. In an embodiment, the binding partner is a receptor, channel, or other complex present on the surface of a target cell. These additional TfR1 binding moieties can target, e.g., specific cell types or cell states within the set of target cells targeted by the n-mer motif. As used herein, "cell state" describes transient elements of a cell's identity. Cell state can be considered a cell's transient characteristic profile or phenotype. Cell states arise transiently during time-dependent processes, either in a unidirectional temporal progression (e.g., during differentiation or following an environmental stimulus) or in a state vacillation that is not necessarily unidirectional and in which the cell may return to the origin state. Vacillating processes can be oscillatory (e.g., cell-cycle or circadian rhythm) or transition between states with no predefined order (e.g., due to stochastic or environmentally controlled molecular events). These time-dependent processes may occur transiently within a stable cell type (as in a transient environmental response) or lead to a new, distinct type (as in differentiation). See, e.g., Wagner et al., 2016. Nat Biotechnol. 34(11): 1145-1160.

[0068] In an embodiment, the additional TfR1 binding moiety is, or includes, a peptide or a polypeptide. In an embodiment, the additional TfR1 binding moiety is, or includes, an antibody or fragment thereof. Exemplary antibodies and fragments are described in greater detail elsewhere herein, e.g., in the discussion on exemplary cargos. In an embodiment, the additional TfR1 binding moiety is or includes an aptamer. In an embodiment, the additional TfR1 binding moiety is or includes a small molecule. In an embodiment, the additional TfR1 binding moiety is or includes a nucleic acid (e.g., DNA or RNA). In an embodiment, the additional TfR1 binding moiety is or includes a receptor. In an embodiment, the additional TfR1 binding moiety is or includes a receptor ligand. In an embodiment, the additional TfR1 binding moiety is or includes a carbohydrate (e.g., a sugar). In an embodiment, the additional TfR1 binding moiety is or includes a lipid. In an embodiment, the additional TfR1 binding moiety is an engineered protein scaffold. In an embodiment, the additional TfR1 binding moiety is an affibody. In an embodiment, the additional TfR1 binding moiety is an antibody mimetic. In an embodiment, the additional TfR1 binding moiety is an engineered binding protein, such as 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 et al., Nat. Biotechnol. (1997) 15(8):772- 777). In example embodiments, the additional TfR1 binding moiety is a receptor ligand or binding protein. In an embodiment, the additional TfR1 binding moiety is attached or otherwise coupled to the capsid surface. In an embodiment, the additional TfR1 binding moiety is encoded by a vector that produces a capsid of the present invention described herein.Engineered Recombinant AAV Genomes for Expression of GBA1

[0069] A transgene encoding GBA1 is inserted into a recombinant AAV genome flanked by AAV ITR sequences and operably linked to a regulatory element that promotes the expression of GBA1 in the CNS. In an embodiment, the engineered AAV capsid encoding polynucleotide can be operably coupled to a poly-adenylation tail. In an embodiment, the poly-adenylation tail can be an SV40 poly-adenylation tail.GBA1

[0070] GBAI, also called glucosylceramidase beta 1; GBA, GCB; and GLUC, is located on the human 1q22 locus and encodes lysosomal membrane proteins. In an embodiment, the polynucleotide sequence in the recombinant AAV genome is a DNA sequence derived from the primary accession number P04062. In another example embodiment, the DNA sequence is P04062. In another example embodiment, the DNA sequence is derived from the secondary accession numbers A8K796, B7Z5G2, B7Z6S1, J3KQG4, J3KQK9, Q16545, Q4VX22, Q6I9R6, and Q9UMJ8. In another example embodiment, the DNA sequence is selected from the group consisting of A8K796, B7Z5G2, B7Z6S1, J3KQG4, J3KQK9, Q16545, Q4VX22, Q6I9R6, and Q9UMJ8.

[0071] In another example embodiment, the polynucleotide sequence included in the vector encodes an RNA sequence derived from: NM_000157.4; NM_001005741.3; NM_001005742.3; NM_001171811.2; and NM_001171812.2. In another example embodiment, the polynucleotide sequence included in the vector encodes an RNA sequence selected from the group consisting of NM 000157.4; NM 001005741.3, NM 001005742.3, NM 001171811.2; and NM 001171812.2. In another example embodiment, the sequence include in the vector is derived from mRNA selected from the group consisting of: AK291911.1, AK298900.1, AK300186.1, AK300829.1, AK300876.1, AK301374.1, AK301879.1, AK302000.1, AK311242.1, AK312502.1, AL547558.3, AU122729.1, AU131071.1, AU140959.1, AU141794.1, BC003356.1, BI458641.1, BX382279.2, BX477204.1, BX648487.1, D13286.1, DC297079.1, K02920.1, KJ690771.1, KJ690772.1, KJ690773.1, LC050340.1, LC050341.1, LC050342.1, M16328.1, and M19285.1. In another example embodiment, the sequence included in the vector encodes a mRNA sequence selected from the group consisting of: AK291911.1, AK298900.1, AK300186.1, AK300829.1, AK300876.1, AK301374.1, AK301879.1, AK302000.1, AK311242.1, AK312502.1, AL547558.3, AU122729.1, AU131071.1, AU140959.1, AU141794.1, BC003356.1, BI458641.1, BX382279.2, BX477204.1, BX648487.1, D13286.1, DC297079.1, K02920.1, KJ690771.1, KJ690772.1, KJ690773.1, LC050340.1, LC050341.1, LC050342.1, M16328.1, and M19285.1.

[0072] All gene name symbols used throughout the specification refer to the gene as commonly known in the art. The examples described herein that refer to gene names are to be understood to encompass human genes and genes in any other organism (e.g., homologous, orthologous genes). The term homolog may apply to the relationship between genes separated by the event of speciation (e.g., ortholog). Normally, orthologs retain the same function in the course of evolution. Gene symbols may be those referred to by the HUGO Gene Nomenclature Committee (HGNC) or the National Center for Biotechnology Information (NCBI). Any reference to the gene symbol is made to the entire gene or variants. Reference to a gene encompasses the gene product (e.g., protein encoded for by the gene).Regulatory Elements

[0073] Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operably-linked to the nucleic acid sequence to be expressed. In some instances, "recombinant expression vectors" may refer to "recombinant AAV genomes". Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "operably linked" as used herein also refers to the functional relationship and position of a promoter sequence relative to a polynucleotide of interest (e.g., a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence). Typically, an operably linked promoter is contiguous with the sequence of interest. However, enhancers need not be contiguous with the sequence of interest to control its expression. The term "promoter", as used herein, refers to a nucleic acid fragment that functions to control the transcription of one or more polynucleotides, located upstream of the polynucleotide sequence(s), and which is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences including, but not limited to, transcription factor binding sites, repressor, and activator protein binding sites, and any other sequences of nucleotides known in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "tissue-specific" promoter is only active in specific types of differentiated cells or tissues.

[0074] In another embodiment, the vector of the invention further comprises expression control sequences including, but not limited to, appropriate transcription sequences (i.e., initiation, termination, promoter, and enhancer), efficient RNA processing signals (e.g., splicing and polyadenylation (polyA) signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequence), and sequences that enhance protein stability. A significant number of expression control sequences, including promoters that are native, constitutive, inducible, or tissue-specific, are known in the art and may be utilized according to the present invention.

[0075] In another embodiment, the vector of the invention further comprises a post- transcriptional regulatory region. In a preferred embodiment, the post-transcriptional regulatory region is the Woodchuck Hepatitis Virus post-transcriptional region (WPRE) or functional variants and fragments thereof and the PPT-CTS or functional variants and fragments thereof (see, e.g., Zufferey R et al., J. Virol. 1999; 73:2886-2892; and Kappes J, et al., WO 2001 / 044481). In a particular embodiment, the post-transcriptional regulatory region is WPRE. The term "Woodchuck hepatitis virus posttranscriptional regulatory element” or “WPRE,” as used herein, refers to a DNA sequence that, when transcribed, creates a tertiary structure capable of enhancing the expression of a gene (see, e.g., Lee Y, et ah, Exp. Physiol. 2005; 90(1):33-37 and Donello J, et al, J. Virol. 1998; 72(6):5085-5092).

[0076] The term "regulatory element" is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). For example, such regulatory elements are described in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0077] Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as adipose tissue or particular cell types (e.g., adipocytes or adipocyte progenitors). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell-type specific. In an embodiment, a vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Also encompassed by the term "regulatory element" are enhancer elements (e.g., adipose-specific enhancers or Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE)). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to produce transcripts, proteins, or peptides, including fusion proteins or peptides encoded by nucleic acids (e.g., GBA1).

[0078] In an embodiment, the promoter can be constitutive. Suitable tissue-specific and constitutive promoters are discussed elsewhere herein, are generally known in the art, and can be commercially available. Suitable neuronal tissue / cell-specific promoters include but are not limited to, GFAP promoter (astrocytes), SYN1 promoter (neurons), and NSE / RU5' (mature neurons). In an embodiment, the neuronal tissue / cell specific promoters include human elongation factor la-subunit (EFla), shortened fragment of EF1a (EFS), cytomegalovirus (CMV) immediate- early enhancer and / or promoter, chicken p-actin (CBA) and its derivative CAG, glucuronidase (GUSB), or ubiquitin C (UBC), neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), CASI, platelet-derived growth factor B-chain (PDGF-P), CK6, TK, tetracycline- responsive promoter (TRE), phosphoglycerate kinase (PGK), telomerase (hTERT), SV40, intercellular adhesion molecule 2 (ICAM-2), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), P-globin minigene np2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), a cardiovascular promoter (e.g., aMHC, cTnT, and CMV-MLC2k), a liver promoter (e.g., hAAT, TBG), a skeletal muscle promoter (e.g., desmin, MCK, C512) or a fragment, e.g., a truncation, or a functional variant thereof. Reference is made to International Patent Application No WO / 2023 / 081648, hereby incorporated by reference.Polynucleotide Constructs for Generation of rAAV Particles

[0079] The engineered viral capsid and / or capsid proteins can be encoded by one or more engineered viral capsid polynucleotides. In an embodiment, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide or engineered adenovirus capsid polynucleotide. In an embodiment, an engineered viral capsid polynucleotide (e.g., an engineered AAV capsid polynucleotide or engineered adenovirus capsid polynucleotide) can include a 3' polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

[0080] In an embodiment, the engineered polynucleotide can be included in a polynucleotide configured to express the engineered capsid in a host cell system for producing viral particles. The host cell system may also include a construct that expresses a recombinant viral genome that comprises a transgene encoding a polypeptide or nucleic acid operably linked to one or more regulatory sequences that promote expression of the transgene in a target cell, including a recombinant AAV genome where AAV ITR sequences flank the transgene and regulatory sequences.

[0081] In an embodiment, the engineered AAV capsid encoding polynucleotide can be included in a polynucleotide configured to express the engineered capsid in a host cell system for producing AAV viral particles. The host cell system may also include a construct that expresses a recombinant AAV viral genome that comprises a transgene encoding a polypeptide or nucleic acid operably linked to one or more regulatory sequences that promote expression of the transgene in a target cell, including a recombinant AAV genome where AAV ITR sequences flank the transgene and regulatory sequences.Pharmaceutical Formulations

[0082] In some aspects, the techniques described herein relate to a pharmaceutical composition comprising the engineered recombinant AAV particle as described herein and an acceptable carrier. As used herein, “pharmaceutical formulation" refers to combining an active agent, compound, or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. As used herein, “pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is useful in preparing a pharmaceutical formulation that is generally safe, non-toxic, and is neither biologically nor otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable carrier or excipient," as used in the specification and claims, includes one and more than one such carrier or excipient. The compound can optionally be present in the pharmaceutical formulation as a pharmaceutically acceptable salt.

[0083] Suitable pharmaceutically acceptable carriers include but are not limited to, phosphate- buffered saline (PBS) or tris(hydroxymethyl)aminomethane hydrochloride (tris), surfactants such as Pluronic F-68 or Poloxamer 188, sodium chloride magnesium chloride, and / or glycerol.

[0084] In addition, suitable pharmaceutically acceptable carriers may also include water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.

[0085] The pharmaceutical formulations can be sterilized and, if desired, mixed with agents, such as preservatives, stabilizers, salts for influencing osmotic pressure, buffers, and / or surfactants, and the like, which do not deleteriously react with the active compound.

[0086] In an embodiment, the pharmaceutical formulation can also include an effective amount of secondary active agents, including but not limited to biologic agents or molecules, but not limited to, e.g. polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof.METHODS OF DELIVERING GBA1 TO THE CNS

[0087] A method of delivering GBAl to the CNS may comprise administering the rAAV particles described herein or a pharmaceutical formulation to a subject in need thereof. The engineered capsids can deliver a polynucleotide encoding a therapeutic protein or nucleic acid to the CNS, where the protein or nucleic acid is expressed. In an embodiment, the subject suffers from Gaucher's disease. In another embodiment, the subject suffers from or is at risk of developing Parkinson's disease. In an embodiment, Gaucher's disease is Type 2 and Type 3 Gaucher disease. Types 2 and 3 of Gaucher disease are neuronopathic forms of the disorder and affect the central nervous system.

[0088] Compositions of the invention may be formulated for delivery to human subjects, as well as to animals for veterinary purposes (e.g., livestock (cattle, pigs, others)) and other non- human mammalian subjects. The dosage of the formulation can be measured or calculated as viral particles or as genome copies ("GC") / viral genomes ("vg"). Any method known in the art can be used to determine the genome copy (GC) number of the viral compositions of the invention. In an embodiment, the viral compositions can be formulated in dosage units to contain an amount of viral vectors that are in the range of about 1.0 x 10º GC to about 1.0 x 1015 GC (to treat an average subject of 70 kg in body weight), and preferably 1.0 x 1012 GC to 1.0 x 1014 GC for a human patient. Preferably, the dose of virus in the formulation is 1.0 x 10º GC, 5.0 X 10º GC, 1.0 X 1010 GC, 5.0 X 1010 GC, 1.0 X 1011GC, 5.0 X 1011 GC, 1.0 X 1012 GC, 5.0 X 1012 GC, or 1.0 x 1013 GC, 5.0 X 1013 GC, 1.0 X 1014 GC, 5.0 X 1014 GC, or 1.0 x 1015 GC.

[0089] In example embodiments, the dosage is between 0.1 x 1012 vg / kg to 1 x 1014 vg / kg. In example embodiments, the therapeutically effective amount is any between 0.1 x 1012 vg / kg to 100 x 1012 vg / kg. In example embodiments, the therapeutically effective amount is any between 1x 1012 vg / kg to 10 x 1012 vg / kg. In example embodiments, the therapeutically effective amount is 5 x 1012 vg / kg.

[0090] In an embodiment, the dosage can be 1X10¹ particles per pL, nL, µL, mL, or L to 1X1020 / particles per pL, nL, µL, mL, or L or more, such as about 1X10¹, 1X102, 1X103, 1X104, 1X105, 1X106, 1X107, 1X108, 1X10º, 1X1010, 1X1011, 1X1012, 1X1013, 1X1014, 1X1015, 1X1016, 1X1017, 1X1018, 1X1019, to / or about 1X1020 particles per pL, nL, µL, mL, or L. In an embodiment, the effective titer can be about 1X10¹ transforming units per pL, nL, µL, mL, or L to 1X1020 / transforming units per pL, nL, µL, mL, or L or more, such as about 1X10¹, 1X102, 1X103, 1X104, 1X105, 1X106, 1X107, 1X108, 1X10º, 1X1010, 1X1011, 1X1012, 1X1013, 1X1014, 1X1015, 1X1016, 1X1017, 1X1018, 1X1019, to / or about 1X1020 transforming units per pL, nL, µL, mL, or L. In an embodiment, the MOI of the pharmaceutical formulation can range from about 0.1 to 10 or more, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 or more.

[0091] The viral vectors can be formulated conventionally using one or more physiologically acceptable carriers or excipients. The viral vectors may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injection formulation may be presented in unit dosage form (e.g., in ampoules or multi-dose containers) with an added preservative. The viral compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing, or dispersing agents. Liquid preparations of the viral vector formulations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g. lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts. Alternatively, the compositions may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0092] The rAAV particles or pharmaceutical formulations thereof described herein can be administered one or more times hourly, daily, monthly, or yearly (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times hourly, daily, monthly, or yearly). In an embodiment, the pharmaceutical formulations or dosage forms thereof described herein can be administered continuously over a period of time ranging from minutes to hours to days. Devices and dosages forms are known in the art and described herein that are effective to provide continuous administration of the pharmaceutical formulations described herein. In an embodiment, the first one or a few initial amount(s) administered can be a higher dose than subsequent doses. This is typically referred to in the art as a loading dose or doses and a maintenance dose, respectively. In an embodiment, the pharmaceutical formulations can be administered such that the doses over time are tapered (increased or decreased) overtime so as to wean a subject gradually off of a pharmaceutical formulation or gradually introduce a subject to the pharmaceutical formulation.

[0093] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLESExample 1 - An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene deliveryMain Text:

[0094] A critical challenge in the development of more efficient delivery vectors for gene therapy is engineering vectors with known mechanisms of action (MOAs) present in human patients. Conventional approaches to engineering vectors such as adeno-associated viruses (AAVs) with enhanced tropisms have largely relied on capsid library selections in animals. These selections have been used by numerous groups over the past two decades because they can be successful without requiring prior knowledge of an MOA (1-7). However, vectors selected in animals mostly have not translated across preclinical models. Despite extensive searches, AAV capsids with clear translational potential and known MOAs for entering the CNS have not yet emerged. Here, Applicants approached this challenge from a different perspective and selected AAV capsids first for a specific MOA, that is binding to the human Transferrin Receptor (TfR1), and showed that these capsids crossed the blood-brain barrier (BBB) and mediated efficient gene delivery throughout the CNS.

[0095] Applicant chose TfR1 as a target because of its high expression on the human BBB; its ability to mediate constitutive, ligand-independent receptor-mediated transcytosis (RMT) across the CNS vasculature (8-12); and its track record as a target to increase the delivery of biologics into the CNS of mice (13-15), nonhuman primates (NHPs) (16–18), and humans as investigational therapies (19-22) and an approved antibody-based therapeutic for Mucopolysaccharidosis type II (23). A 7-mer-modified AAV9 capsid libraries were screened for its ability to bind to human TfR1 in vitro. Capsids were selected that exhibited more efficient gene delivery to human brain endothelial cells and improved active transport across a human vascular monolayer. When systemically administered to adult TFRC knock-in (KI) mice carrying a chimeric Tfrc gene with a humanized extracellular domain, BI-hTFR1 transduced the majority of neurons and astrocytes across multiple brain regions. The enhanced tropism depended entirely on an interaction with the humanized TfR1, as Applicants observed no enhancement in wild-type (WT) mice. The tropism was also selectively enhanced in the CNS, consistent with the high level of expression of TFRC on the CNS vasculature relative to the vasculature in other organs (12).

[0096] To explore its ability to deliver a therapeutically relevant cargo, Applicants intravenously injected TFRC KI mice with an example capsid, BI-hTFR1 or AAV9, packaging the human Glucosylceramidase Beta 1 (GBA1) gene. Inactivating mutations of GBAI are the primary cause of Gaucher's disease, a lysosomal storage disorder often affecting the CNS, and GBA1 mutations are a genetic risk factor for Parkinson's disease and dementia with Lewy Bodies (24). Systemic delivery of BI-hTFR1:GBA1 in adult TFRC KI mice resulted in robust expression throughout the brain and increased glucocerebrosidase (GCase) activity in the brain and cerebrospinal fluid (CSF), which were all not observed with AAV9. Through its direct engagement of TfR1, a transporter of natural protein ligands and therapeutic biologics in humans, BI-hTFR1 represents a promising vector for developing CNS-targeting human gene therapies.ResultsAAV capsids engineered to bind human TfR1

[0097] To target AAV capsids to TfR1, Applicants screened AAV9-based NNK capsid libraries of variants with random 7-mer insertions between VP1 residues 588-589 for selective binding to human TfR1 using recently described receptor-targeting approach (25) (FIG. 1A). Based on the results of the in vitro screens, Applicants chose four capsids that share a common sequence motif for validation: BI-hTFR1, BI-hTFR1-2, BI-hTFR1-3, and BI-hTFR1-4. Applicants used each capsid to individually package a single-stranded genome that expresses nuclear mScarlet and Luciferase under the control of the ubiquitous CAG promoter (ssAAV-CAG-NLS-mScarlet- P2A-Luciferase-WPRE-SV40pA). The four capsids exhibited elevated association with and transduction of CHO cells that stably express human TfR1 but not the control CHO cells or those that express TfR1 from rhesus macaque, marmoset, or mouse (FIG. 1B and C, and FIG. 7). The capsids were tested for increased association with and transduction of human brain endothelial cells that endogenously express TFRC. Although the extent of AAV associated with primary human brain microvascular endothelial cells (hBMVECs) and a human brain endothelial cell line (hCMEC / D3) varied (FIG. 1D), each of the four TfR1-binding capsids exhibited significantly enhanced transduction of both brain endothelial cell types compared to AAV9 (FIG. 1E). Το further assess the relative binding affinities of the capsids with purified human TfR1, Applicants performed bio-layer interferometry (BLI). Kinetic constants using a multi-phasic binding model were derived because a monovalent interaction algorithm led to a poor data fit. Factors that may contribute to the multi-phasic binding curves are outlined in the Materials and Methods section below. Based on its performance across cell association and transduction assays and its ability to bind human TfR1 at lower concentrations, Applicants chose BI-hTFR1 as an example of rAAV for further investigation.BI-hTFR1 binds the TfR1 apical domain

[0098] To evaluate whether binding to TfR1 is necessary for the increased transduction of brain endothelial cells by BI-hTFR1, Applicants assessed hCMEC / D3 transduction in the presence of two anti-TfR1 antibodies: OKT9, a monoclonal antibody that binds the TfR1 apical domain (26) or AF2474, a polyclonal antibody that competes with Transferrin (Tf) for TfR1 binding. Transduction was significantly inhibited by increasing concentrations of OKT9 but not by AF2474 (FIG. 2A). This suggests that an interaction with the human TfR1 apical domain was required for the enhanced transduction mediated by BI-hTFR1 in these cells. Consistent with this result, OKT9 directly competed with BI-hTFR1 for binding to purified human TfR1 (FIG. 8B). Notably, binding to the TfR1 apical domain is a common feature of several antibody-based BBB shuttles that have been tested in humans and likely positions the binding site away from the Tf binding site. This is important because surface TfR1 is thought to be occupied mainly by Tf based on the typical 10- 20 µM blood concentration of iron-bound holo-Tf (27) and its sub-nanomolar binding affinity for TfR1 (FIG. 8C). Given the double-digit nanomolar affinity of BI-hTFR1 for human TfR1, Tf would be predicted to outcompete BI-hTFR1 for binding to TfR1 if their binding epitopes overlap. Using BLI, Applicants found that BI-hTFR1 binds human TfR1 with similar kinetics, whether in the presence or absence of a receptor-saturating concentration of holo-Tf (FIG. 2B and FIG. 8D), and that BI-hTFR1 is capable of binding a preformed complex of human TfR1 and Tf (FIG. 2C). Consistent with the BLI results, OKT9, but not Tf, inhibited the association of BI-hTFR1 with hCMEC / D3 cells and its colocalization with human TfR1 (FIG. 2D and FIG. 9).Active trafficking of BI-hTFR1 across a human BBB model

[0099] Applicants established a BBB transwell model using the hCMEC / D3 cell line to test AAV transport across a human endothelial cell barrier. They compared the amount of BI-hTFR1 transported across the endothelial barrier relative to AAV9 and AAV2. To minimize the effect of well-to-well variation, Applicants mixed BI-hTFR1, AAV9, and AAV2 carrying barcoded genomes that were individually identifiable by qPCR (table S1), added this mixture to the media of the upper chambers, which were maintained at 37°C or 4°C, and measured transport to the lower chamber three hours later (FIG. 3A). At 4°C, ATP-dependent transcytosis is suppressed but passive transport, at least in part due to paracellular crossing, can occur, providing a measurement of leakiness that is a known issue with the use of hCMEC / D3 transwell assays (28). As previously reported, AAV9 crossed the transwell barrier more efficiently than AAV2 at 37°C, and the number of vector genomes detected in the bottom chamber was significantly reduced at 4°C for both AAVs as is consistent with ATP-dependent transcytosis (29-31). Significantly more BI-hTFR1 was actively transported to the bottom chamber than AAV9 or AAV2 (FIG. 3B). Passive transport of BI-hTFR1 and AAV2 was reduced relative to AAV9, likely due to increased association with the cells.

[0100] RMT involves endocytosis, vesicular trafficking, and exocytosis (32). Whether BI- hTFR1 transcytoses hCMEC / D3 cells via the RMT pathway of TfR1 was explored by assessing its colocalization with various organelle markers. BI-hTFR1 partially colocalized with markers of the early and late endosomal pathway (Rab5 and Rab7) as well as the trans-Golgi network (TGN46), but did not extensively colocalize with markers of the cis-Golgi (Rcas1) or endoplasmic reticulum (KDEL) (FIG. 3C and FIG. 10). In contrast, AAV2 was not highly colocalized with any of the endosomal or organelle markers except TGN46. Partial colocalization of both AAVs with TGN46 is consistent with the requirement for trafficking AAVs to the trans-Golgi for transduction (33). Cell surface TfR1 clustering promotes clathrin-coated pit formation (34) and uptake into a subpopulation of endosomes enriched in Rab5 (35). This is consistent with the observation that BI-hTFR1 colocalized with Rab5 to a greater degree than AAV2. A smaller proportion of TfR1 colocalizes with Rab7 (36), which decorates endosomes associated with transcytosis (37) and the lysosomal degradation pathway. This is again consistent with the observation of a partial overlap between BI-hTFR1 and Rab7 localization in the cells. The colocalization assays suggest that BI- hTFR1 engages TfR1 and is actively transported across the human BBB model via RMT. In contrast, AAV2 is predominantly trafficked to the trans-Golgi (33).CNS tropism of BI-hTFR1

[0101] Having established that BI-hTFR1 is more effective at transducing human brain endothelial cells via its interaction with TfR1 and is more efficiently transported across a human BBB model, Applicants next sought to investigate whether BI-hTFR1 could cross the BBB in vivo by engaging human TfR1. To test this, Applicants used TFRC knock-in (KI) C57BL / 6J mice in which exons 4–19 of the mouse Tfrc encoding the extracellular domain were replaced by the corresponding region of human TFRC (FIG. 4A) (38). Levels of the mouse-human hybrid gene mRNA and protein products were similar to those in WT mice (FIG. 11) and are suitable for assessing the tropism of AAVs targeted to human TfR1. BI-hTFR1 or AAV9:CAG-NLS- mScarlet-P2A-Luciferase-WPRE-SV40pA was intravenously injected at 5 x 1011 vg / mouse into adult female C57BL / 6J_or_TFRC KI mice (FIG. 4B). Three weeks post-injection, Applicants observed enhanced biodistribution to and transduction of the brain and spinal cord by BI-hTFR1 in the TFRC KI but not in control C57BL / 6J mice (FIG. 4C and D, and FIG. 12). Increased biodistribution and transduction were not observed in any of the other organs that were assessed, indicating that the interaction with human TfR1 selectively enhanced the CNS tropism. Comparing the TFRC KI mice that received BI-hTFR1 to the C57BL / 6J mice that received AAV9, Applicants detected 6-fold and 12-fold more viral genomes; 54-fold and 43-fold higher viral mRNA transcript levels; 132-fold and 58-fold greater Luciferase enzymatic activity in the brain and spinal cord, respectively. As expected, AAV9 transduction was not significantly affected by genotype in any of the assessed organs.

[0102] To evaluate the efficiency of transduction of specific cell types within the CNS, Applicants assessed the fraction of cells positive for markers of neurons or astrocytes that expressed mScarlet. In TFRC KI mice, BI-hTFR1 transduced a mean of 54, 32, and 68% of NeuN+ neurons in the cortex, striatum, and thalamus, respectively (FIG. 5C and D). In contrast, in C57BL / 6J mice, BI-hTFR1 transduced less than 1% of NeuN+ cells in the cortex striatum and thalamus. AAV9 transduced 1-6% of NeuN+ cells in these same regions regardless of the mouse strain. BI-hTFR1 transduced more than 80% of the SOX9+ astrocytes in the cortex, striatum, and thalamus of TFRC KI mice, in comparison with 10% or fewer SOX9+ cells in C57BL / 6J mice or 20% or fewer SOX9+ cells transduced by AAV9 in C57BL / 6J mice (FIG. 5E and 5F). Consistent with the biodistribution and transduction data, mScarlet levels in the livers of C57BL / 6J and TFRC KI mice were comparable between AAV9 and BI-hTFR1 (FIG. 12A and 12B). A significant reduction of biodistribution to the DRGs in TFRC KI animals treated with BI-hTFR1 relative to AAV9 controls (FIG. 4C) was observed; DRG cell transduction by AAV9 or BI-hTFR1 in C57BL / 6J or TFRC KI mice is shown in FIG. 12C.BI-hTFR1 mediates GBA expression throughout the brain

[0103] BI-hTFR1's ability to deliver the human gene Glucosylceramidase Beta 1 (GBAI) to TFRC KI mice was evaluated. A single-stranded AAV genome was built containing a hybrid cytomegalovirus enhancer-chicken beta-Actin (CMV-CBA) promoter driving the expression of human GBAI with a C-terminal influenza virus hemagglutinin (HA) tag (FIG. 6A) (39). The genome was packaged into AAV9 or BI-hTFR1 and administered intravenously to TFRC KI mice at a dose of 1 x 1014 vg / kg, which is comparable to the weight-adjusted dose of the FDA-approved AAV9-based Zolgensma gene therapy for spinal muscular atrophy. Additionally, Applicants administered BI-hTFR1:GBA1 to a separate group of TFRC KI mice at a 20-fold lower dose (5 x 1012 vg / kg). For the untreated control mice, Applicants used C57BL / 6J mice, which possess the same genetic background of the TFRC KI mice. Three weeks post-administration, Applicants detected ~30 times more AAV genomes in the brains of mice treated with BI-hTFR1 as compared with those of mice that received AAV9 (FIG. 6B). We observed robust brain-wide expression of GBA-HA as assessed by HA immunostaining (FIG. 6C) and significantly elevated glucocerebrosidase (GCase) activity in brain tissue lysates and the CSF, compared to the mice injected with the high dose of AAV9 or control C57BL / 6J mice (FIG. 6D, and FIG. 13A and 13B). In the mice injected with the high dose of AAV9, Applicants observed minimal GBA expression in the brain but similar amounts of viral genomes, GBA expression, and GCase activity in the liver compared to the mice injected with the high dose of BI-hTFR1 (FIG. 6B and 6D, and FIG. 13C); as expected, in the mice injected with the low dose of BI-hTFR1, the amount of viral genomes in the liver was reduced relative to the high dose AAV9 group. Reductions in GCase activity in the brain and CSF have been reported to be associated with the progression of Parkinson's disease (40, 41), and previous studies suggest that GBAI delivery to the brain can treat neuronal a- synucleinopathy (42–44) Notably, in the mice that received the low dose of BI-hTFR1, Applicants observed neuronal transduction in regions linked to motor symptoms of Parkinson's disease such as the substantia nigra pars compacta, deep cerebellar nuclei, and other parts of the brainstem (FIG. 6C and Data FIG. 13A) (45). Under the same imaging conditions, GBA-HA was not detected in the brains of the mice injected with the high dose of AAV9. This suggests that a 20-fold lower dose of BI-hTFR1 led to greater GBA levels in disease-relevant brain regions compared to AAV9.Discussion

[0104] In this study, Applicants leveraged engagement of the apical domain of human TfR1- to shuttle an engineered AAV capsid, BI-hTFR1, across the BBB. Protein shuttles designed to bind human TfR1 to facilitate RMT are now in the clinic and are showing evidence of CNS entry (NCT04023994) and efficacy (46). The TfR1 apical domain is also a receptor target of natural viruses (47-51), indicating that this iron transporter can be exploited for viral entry. Importantly, Applicants demonstrated that BI-hTFR1 binds human TfR1 and pre-formed TfR1-Tf complexes with comparable kinetics and does not compete with holo-Tf for TfR1 binding. The ability to bind TfR1 in the presence of its natural ligand is critical because AAVs cannot be administered at doses capable of competing with Tf for TfR1 binding. The use of this validated transport mechanism bolsters Applicants confidence that BI-hTFR1 can perform well as a CNS-targeting human gene therapy vehicle.

[0105] BI-hTFR1 was engineered to bind TfR1 by inserting a novel 7-mer TfR1 recognition sequence directly within surface loop VIII of the capsid. Because the approach did not require the introduction or conjugation of bulky protein domains as a means to retarget the AAV (52–58), Applicants do not anticipate additional complexities in the manufacturing of BI-hTFR1 compared to AAV9, one of the top producing natural AAVs. Indeed, Applicants consistently produced and purified BI-hTFR1 with final yields within twofold of AAV9 (Table S2).

[0106] As compared to natural AAV capsids such as AAV9 and AAV2 that are used in approved gene therapies, BI-hTFR1 demonstrated the remarkable ability to be actively transported across a human brain endothelial cell layer and to cross the BBB and transduce cells throughout the brains and spinal cords of TFRC KI mice after intravenous delivery. BI-hTFR1 exhibited a broad CNS tropism enhancement similar to those observed with engineered mouse BBB-crossing capsids, e.g., AAV-PHP.B, BI28, AAVF, and 9P31 that bind to mouse GPI-anchored proteins LY6A, LY6C1, or Car4 (25, 59, 60). By engaging highly abundant CNS endothelial cell surface proteins as new receptors, these capsids are able to efficiently cross the BBB and gain access to parenchymal cells.

[0107] A critical advantage of BI-hTFR1 and other capsids developed through target receptor binding screens is that their MOA is well established, thus their species tropism is predictable and can inform large animal experiments. This stands in contrast to capsids identified from conventional selections with unknown MOAs and therefore unknown potential to translate to other species including humans. Human TfR1-targeting AAVs reported in this study is not expected to exhibit enhanced CNS tropisms in NHPs such as macaques and marmosets. Therefore, tests in these species would only be valuable in terms of profiling biodistribution, immune responses, and toxicity in the absence of the target, human TfR1. Knowing the precise target of the AAV capsid also aids the prediction of its function in human patients. For instance, there are no known missense variants with an allele frequency greater than 1 in a 1000 within the TFRC apical domain (61). Therefore, Applicants predict that BI-hTFR1 binding to human TfR1 will not be impacted by common variations within the coding sequence of the human apical domain.

[0108] A second advantage of human receptor-targeting capsids is that gene therapies leveraging these vectors can be tested in KI mouse models possessing both the human target receptor and disease-relevant mutation(s). For instance, disease models are typically created using mice and, in the absence of a capsid with an enhanced CNS tropism that translates across species, studies will need to use a surrogate mouse BBB-crossing capsid to deliver and test their potentially therapeutic payloads in these disease models. With BI-hTFR1, the gene therapy product that would be delivered to human patients can be tested, without switching out the capsid for a surrogate, in a mouse disease model that has been crossed with TFRC KI mice. Using a therapeutically relevant payload, Applicants found that BI-hTFR1 could deliver 30 times the number of AAV:GBA1 genomes to the brains of TFRC KI mice and increase brain and CSF GCase activity as compared to AAV9. Even at a 20-fold lower dose, GBAI expression was detectable throughout the brain in BI-hTFR1:GBAI treated mice. However, the TFRC KI mice were WT with respect to Gbal and only the high dose of BI-hTFR1:GBA1 led to increased brain and CSF GCase activity that were detectable above the endogenous mouse GCase activity.

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Huang, An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice. J. Vis. Exp. (2018), doi:10.3791 / 56774.Supplementary Materials for Example 1Materials and MethodsAnimals

[0110] All procedures were performed as approved by the Broad Institute IACUC. C57BL / 6J (000664) were obtained from The Jackson Laboratory (JAX). TFRC KI mice (C57BL / 6- Tfr1tm1TFR1 / Bcgen, B-hTFR1, 110861) were obtained from Biocytogen. Recombinant AAV vectors were administered intravenously via the retro-orbital sinus in young adult male or female mice. Mice were randomly assigned to groups based on predetermined sample sizes. Transgenic animals were genotyped using DNeasy Blood & Tissue Kit (Qiagen, 69504) using primers listed in table S1. Two mice were excluded from analysis in FIG. 4, FIG. 5, FIG. 11, and FIG. 12: The first was a mouse assigned to the AAV9 in the TFRC KI group but was determined to be a WT by genotyping and western blotting. One mouse in the BI-hTFR1-treated C57BL / 6J group displayed no evidence of transduction in the biodistribution, transduction, or Luciferase assays, and showed no fluorescent reporter signal in the liver or the brain. Experimenters were not blinded to the sample groups.

[0111] Mice were euthanized with Euthasol and transcardially perfused with PBS at room temperature followed by 4% paraformaldehyde (PFA) in ice cold PBS. Tissues were post-fixed overnight in 4% PFA in PBS and sectioned by vibratome. Immunohistochemistry (IHC) was performed on floating sections with antibodies diluted in PBS containing 5% donkey serum, 0.1% Triton X-100, and 0.05% sodium azide. Primary antibodies NeuN 1:500 (Invitrogen, MA5-33103), SOX9 1:250 (abcam, ab185966) were incubated at room temperature overnight. The sections were then washed and stained with secondary Alexa-conjugated antibodies Alexa Fluor 647 (Invitrogen, A-31573) and Alexa Fluor 555 (Invitrogen, A-21427) at 1:1000 for four hours or overnight.Plasmids

[0112] AAV9 and the BI-hTFR1 Rep-Cap plasmids were generated by gene synthesis (GenScript). The CAG-WPRE-hGH pA backbone was obtained from Viviana Gradinaru's lab through Addgene (#99122). GFP, GFP-2A-luciferase, and mScarlet cDNAs were synthesized as gBlocks (IDT) or synthesized and cloned (GenScript).

[0113] Full length TFRC cDNA expression and lentiviral plasmids were cloned by inserting the open reading frames of TFRC (human: NM_003234.4; mouse: NM_011638.4; marmoset: NM_001301847.1, macaque: NM_001257303.1) into the lentiviral backbone pLenti-EF-FH-TAZ- ires-blast (Addgene #52083) with EcoRI / Sall sites.

[0114] For the C-terminal Fc fusion protein, the coding sequence of the extracellular region of human TfR1 (residues 89-760) were amplified by PCR and inserted into pCMV6-XL4 FLAG- NGRN-Fc (Addgene #115773) with EcoRV and Xbal sites. The signal peptide H7 (ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTTAGAGGTGTCCAGTGT (SEQ ID NO: 43843) was introduced to N terminal of the TFRC sequence to direct protein secretion.

[0115] For the N-terminal Fc fusion protein, the coding sequence of the extracellular region of human TfR1 (residues 89-760) was amplified by PCR. An N-terminal Fc region with an N- terminal H7 signal peptide and a C-terminal GS linker was ordered from IDT. The desired region of plasmid PCMV6-XL4 FLAG-NGRN-Fc (Addgene #115773) was amplified by PCR. These three DNA fragments were assembled with NEBuilder® HiFi DNA Assembly (NEB E2621).Cell lines and primary cultures

[0116] HEK293T / 17 (CRL-11268), Pro5 (CRL-1781), and CHO-K1 (CCL-61) were obtained from ATCC. hBMVECs cells (H-6023) were obtained from Cell Biologics and cultured as directed by the manufacturer. hCMEC / D3 cells were obtained from Millipore-Sigma (SCC066).Reconstitution of full-length TfR1

[0117] Full length human TfR1 was reconstituted into Peptidiscs (PEPTIDISC LAB, dissolved at 2 mg / mL in 20 mM TrisHCL, pH 8). Briefly, HEK293 cells expressing C-terminally tagged full length human TfR1 were harvested by centrifugation at 300 × g. Cells were lysed by sonication and fractionated by centrifugation at 15,000 × g to remove nuclei and cell debris and at 100,000 × g to pellet the cell membrane. Membrane was resuspended in Buffer A (TrisHCl, pH 7.5, 50 mM NaCl, 10% glycerol) to 10 mg / mL, and then supplemented with 0.8% n-Dodecyl-ẞ-D-Maltoside (DDM, Goldbio, no. DDM5) at 4°C with gentle agitation to solubilize the membranes. Anti-FLAG M2 magnetic beads (Sigma-Aldrich) were incubated with the solubilized membrane and washed with buffer A with 0.02% DDM. Beads were incubated for five minutes at room temperature in a solution of 1 mg / mL Peptidisc peptide and then thoroughly washed in buffer A without detergent. Peptidisc-TfR1 was eluted in TrisHCl, pH 7.5, 150 mM NaCl, 0.1 mg / mL 3XFLAG peptide and analyzed by SDS-PAGE.Bio-Layer Interferometry

[0118] A GatorPrime instrument was used for the characterization of the binding dynamics for all BLI studies and all analysis was conducted with Gator Bio software v 2.10.4.0713. Experiments were run in black 384 well plates (Greiner Bio-One, 781906). All reagents and buffer wells were prepared with Q buffer and probes from Gator Bio.

[0119] For AAV capsid binding to full length human TfR1, AAVx probes (Gator Bio) were equilibrated in Q buffer and then loaded with either BI-hTFR1, BI-hTFR1-2, BI-hTFR1-3, BI- hTFR1-4, or AAV9 by uniform thresholding to an 8 nm shift. Seven serial dilutions of full length human TfR1-FLAG were associated to each capsid for 120 seconds and then dissociated in Q buffer for 120 seconds. Only the five highest concentrations are shown on sensorgrams. Three trials of the assay were performed. A probe incubated with Q buffer instead of human TfR1 was included in each trial for background subtraction.

[0120] For BI-hTFR1 and AAV9 binding to pre-incubated Tf with full length human TfR1, assays and analysis were conducted as above but with 300 nM holo-Tf (Millipore Sigma T413) pre-incubated with each dilution of TfR1 for 15 minutes.

[0121] For kinetic analysis of capsid binding to TfR1, the sensorgram data from each of three trials using seven different analyte concentrations per capsid displayed clear multi-phasic binding and dissociation characteristics that could not be explained by a monovalent interaction. Therefore, Applicants used the global 2:1 exponential association and decay model. However, Applicants are not implying that the binding between the TfR1-targeting capsids and TfR1 is bivalent. The kinetic constants shown in FIG. 8 should be interpreted with caution due to the following factors: First and foremost, the high density of binding epitopes on the AAV surface may lead to local increases in the effective concentration of the receptor analyte, which results in a higher apparent affinity(62). Second, TfR1 itself is bivalent, which could contribute to the aforementioned avidity artifact and also result in the bridging of closely loaded adjacent AAVs. While probes were loaded to less than 60% of their total binding capacity, the uniformity of loading cannot be controlled and a fraction of the measurements may result from 2:1 binding of the receptor dimers to adjacent capsids(63). Third, transitional binding states due to conformational changes or sequential binding events may contribute to the observation of multi-phasic binding kinetics.

[0122] The software-computed rate constants from these replicate fittings were subject to QC thresholding that included R2 > 0.99, Gator-computed error ratios of <25% of their respective kinetic constants, and fitting residuals <10%. In the cases of both BI-hTFR1-3 and BI-hTFR1-4, one replicate was eliminated due to a kon error ratio of greater than 25%. Trials with QC threshold- passing data were averaged and their standard error was taken.

[0123] For the tandem competition binding assays, a human TfR1 apical domain-targeting antibody, OKT9 (Thermo Fisher Scientific) was chosen as a competitor. The extracellular domain of human TfR1 (residues 89–760) was recombinantly expressed as an N-terminal Fc-fusion protein (Fc-hTfR1) in Expi293 suspension cells and purified by gravity flow with PierceTM Protein A Agarose (Thermo Fisher Scientific). HFCII probes were loaded with Fc-hTfR1 by thresholding to a 1 nm shift. Fc-TfR1-loaded probes were associated in tandem, first to BI-hTFR1 until the signal reached approximately 2 nm, and subsequently to the OKT9 antibody at 250, 100, 50, or 25 nM or in Q buffer alone. The latter was included to demonstrate BI-hTFR1 dissociation in the absence of competitor OKT9. In parallel, a Fc-hTfR1 loaded probe was associated first in Q buffer alone and next in 250 nM of the OKT9 antibody to demonstrate maximum antibody binding in the absence of capsid. The two association phases were 240 and 360 seconds, respectively.

[0124] For the triple tandem binding assays involving holo-Tf, holo-Tf was biotinylated for two hours on ice with an EZ-LinkTM Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher Scientific) and then buffer exchanged into PBS with a 0.5 mL Pierce Protein Concentrator PES column with a 10K MWCO to remove unreacted biotin. Streptavidin (SA) probes were equilibrated in Q buffer and then loaded to a 2 nm shift with biotinylated holo-Tf or were incubated in Q buffer to serve as a no-ligand control. Three of the probes were submitted to sequential binding steps for 240 seconds each. The probes were incubated in Q buffer for 240 or 120 seconds between each binding step to demonstrate <10% baseline drift (i. e., loss of binding) before binding the next molecule. In channel 1, the holo-Tf-loaded probe was associated first with 150 nM full length human TfR1, next with Q buffer only, and finally with BI-hTFR1. In channel 2, the holo-Tf loaded probe was associated with TfR1, then 200 nM OKT9 antibody, and finally with BI-hTFR1 capsid. In channel 3, the holo-Tf-loaded probe was associated with Q buffer alone, then the OKT9 antibody, and finally with BI-hTFR1. In channel 4, the unloaded probe was associated with TfR1, then OKT9 antibody, and finally with BI-hTFR1 to reveal any nonspecific binding to the probe.

[0125] For full length hTfR binding to human holo-Tf, MFC probes were equilibrated in Q buffer and then loaded with anti-Tf monoclonal antibody 12A6 (Thermo Fisher Scientific) to a 1 nm shift. Next, holo-Tf was loaded as ligand to saturation. Serial dilutions of full length peptidisc- reconstituted hTfR1-FLAG were associated with holo-Tf for 120 seconds and then dissociated in Q buffer for 120 seconds. Three trials of the experiment were performed. A probe incubated with Q buffer alone was included in each trial for background subtraction in sensorgram curve fittings and kinetics analysis. For subsequent kinetics analysis, the sensorgram data from each of three trials were fitted with global 1:1 exponential association and decay models. The software- computed rate constants from these replicate fittings were subject to QC thresholding that included R2 > 0.99, computed error ratios of <10% of their respective kinetic constants, and fitting residuals <10%. Kinetics values were averaged and their standard error was taken.Lentivirus production

[0126] Lentivirus was produced with a third-generation lentivirus system by cotransfection of three packaging plasmids (pMDLg-RRE, pRSV-Rev, and pVSV-G) and vector plasmid (pLenti- EF-FH-TAZ-IRES-blast, a gift from Yutaka Hata, Addgene plasmid # 52083) encoding the entire coding sequences of TFRC or Tfre (human: NM_001128148; macaque: NM_001257303; marmoset: NM_001301847; or mouse: NM_011638.4) at a ratio of 4:1:1:6 in HEK293T / 17 cells. Lentiviruses were harvested from the media three days after transfection and filtered with 0.45 µm PES to remove cell debris.Exogenous TfR1 expression in CHO cell lines

[0127] CHO cell lines expressing human TFRC or macaque, marmoset, or mouse Tfrc were established via random lentiviral integration. Briefly, CHO cells were seeded at 150,000 cells / well in 12-well plates and transduced with lentivirus-containing media for 48 hours, then transferred to 10 cm dishes for selection with 10 µg / mL blasticidin for one week followed by maintenance in media with 1 µg / mL blasticidin. To validate expression of the desired construct, RNA was extracted from cells using the Qiagen RNeasy Kit. Maxima H Minus Reverse Transcriptase (ThermoFisher, EP0753) and oligo dT were used for cDNA synthesis. Species-specific primers were used for qPCR (table S1). CHO cells expressing TfR1 from each species were also assessed by immunostaining with two anti-TfR1 antibodies: ab84036 (Abcam) and OKT9 (eBioscienceTM) (FIG. 7).AAV production and titering

[0128] The AAV9 variant capsids carrying CAG-NLS-mScarlet-P2A-Luciferase-WPRE- SV40pA or CMV / CBA-GBA1-HA-pA were either produced in suspension followed by iodixanol purification and titered as previously described (64) or produced using adherent cells as previously described (65). Production, purification methods, and post-purification yields are provided in table S2. For BLI experiments, AAV vectors were produced in adherent HEK293T cells and purified by static binding (3 to 10 plates per preparation). At 72 hours post-transfection, the cells and supernatant (60-200 mL per prep) were collected in 125-500 mL shake flasks, incubated with 0.1% Triton X-100, 2 mM MgCl2 and 25 U / mL benzonase (Sigma, E1014-25KU) for 90 minutes at 37°C, and centrifuged for 10 minutes at 3724 ×g for clarification. The clarified lysates were incubated with 150 µL POROS AAV9 resin (Thermofisher, A27353), at 37°C for 90 minutes, under agitation (1.14 ×g). The mixes were loaded into detergent removal spin columns (Thermofisher, 87778) and supernatants were discarded using a vacuum manifold. POROS AAV9 beads were rinsed three times with 5 mL PBS, using a vacuum manifold. AAV vectors were eluted in 15 mL Falcon tubes, with 2 mL elution buffer (0.1 M Glycine, pH2.5) and neutralized with 500 µL 1 M Tris, 0.5 M NaCl, pH 8. AAVs were dialysed against 3 × 2 L of PBS (with calcium and magnesium) supplemented with 0.001% pluronic F68, using float-a-lyzer G2 dialysis cassettes with a MWCO of 8–10 Kd (Repligen, G235067), or buffer exchanged and concentrated in the same final formulation, using centrifugal filters with a MWCO of 100 Kd (Thermofisher, 88532).Binding assays

[0129] CHO cells stably expressing human TFRC or macaque, marmoset, or mouse Tfrc were seeded at 30,000 cells per well; hCMEC / D3 cells were seeded at 30,000 cells per well; and hBMVEC cells were seeded at 15,000 cells per well. At 24 hours post-seeding, each well was subjected to a media change with the fresh cold media that contained each AAV variant carrying CAG-NLS-mScarlet-P2A-Luciferase-WPRE-SV40pA at 3,000, 6,000, or 12,000 vg / cell for CHO, hCMEC / D3, or hBMVEC cells, respectively. The plate was then maintained at 4°C with gentle rocking for one hour, followed by five phosphate buffered saline (PBS) washes. Cells were treated with Proteinase K at 56°C for one hour, followed by heat inactivation at 95°C for 10 minutes. Total DNA extraction was performed using the DNeasy kit (Qiagen). The DNA was diluted 1:20 and subjected to qPCR with the mScarlet-qPCR-F and mScarlet-qPCR-R primers (table S1).Transduction assays

[0130] CHO cells stably expressing human TFRC or macaque, marmoset, or mouse Tfrc were seeded at 100,000 cells per well; hCMEC / D3 cells were seeded at 10,000 cells per well; and hBMVEC cells were seeded at 5,000 cells per well. At 24 hours post-seeding, each well was subjected to a media change with the fresh media that contained the AAV variant carrying CAG- NLS-mScarlet-P2A-Luciferase-WPRE-SV40pA at 3,000, 6,000, or 12,000 vg / cell for CHO, hCMEC / D3, or hBMVEC cells, respectively, and the plate was maintained at 37°C with 5% CO2 for 24 hours. Transduction was measured using the Britelite Plus reporter gene assay system (PerkinElmer, 6066766). Luciferase activity was reported in relative light units (RLU) as raw data or normalized to AAV9 controls.Antibody inhibition assays

[0131] The hCMEC / D3 cells were seeded at 7,500 cells per well. Two days later, 100 µL of media with 3 x 108 vg / mL_AAV and the specified concentration of OKT9 or R&D AF2474 antibody was transferred to each well. The plate was maintained at 37°C with 5% CO2 for 24 hours. Transduction was measured using the Britelite Plus reporter gene assay system.Transwell assays

[0132] hCMEC / D3 cells (under nine passages) were maintained in EGM2-MV media with VEGF (Lonza, CC4147). Falcon® Permeable Supports for 24-well Plates with 1.0 µm Transparent PET Membranes (Corning, 353104) were prepared by coating with collagen type I (Millipore, 08- 115) diluted 1:50 in PBS and incubating at 37°C for two hours. On Day 0, cells were plated at 30,000 / cm² on the membrane and grown for two days in 200 µL of EGM2-MV media with VEGF on the top of the membrane and 600 µL of media in the lower chamber. On Day 2 and Day 4, the media at both the top and bottom of the transwell was changed to EGM2-MV media without VEGF, and the media at the bottom of the transwell was changed to Astrocyte Conditioned Media (Sciencell, 1811). Starting from Day 4, TEER values were measured using the EVOM2 meter (World Precision Instruments). TEER values were calculated as the ohmmeter readout in wells with cells minus the ohmmeter readout in wells without cells, multiplied by the surface area in cm² units.

[0133] Transwell permeabilities using this process were previously validated by measuring the permeability of Dextran FITC 4k (Sigma, 46944), 40k (Biotium, 76221-470), and 70k (Biotium, 76221-460). TEER values peaked on Day 6, reaching values >10 Ω / cm², which is when AAVs were added to the transwells. BI-hTFR1, AAV2, and AAV9 with capsid-specific barcoded transgenes were pooled and added to the top well of the transwell at 25,000 vg / cell (each AAV). After three hours of incubation at 37°C or 4°C, 22 µL of media was extracted from the bottom chamber. To detect the amount of AAV in the media, extracts were treated with DNasel (NEB, M0303S) for 15 minutes at room temperature, then diluted in TE buffer (5 mM EDTA), and denatured at 70°C for 10 minutes to deactivate DNaseI. DNA extracts were then diluted in 5% Tween 20 with UltraPure™™ Salmon Sperm DNA Solution (ThermoFisher, 15632011). The amount of AAV that passed through the transwell was measured by qPCR using a standard curve of known AAV quantities derived from AAV-containing media applied to the top well.Immunofluorescence staining and colocalization in hCMEC / D3 cells

[0134] For the imaging of BI-hTFR1 or AAV2 binding to hCMEC / D3 cells with or without OKT9 or Tf-647, BI-hTFR1 and AAV2 were added to hCMEC / D3 cells in optical plates (PhenoPlate 96-well, PerkinElmer, 6055302) at 50,000 vg / cell, while Tf-647 (ThermoFisher, T23366) and purified TfR1 antibody (OKT-9, AAT Bioquest, 10713000) at a 1:200 dilution were added to wells to reach a final concentration of 1 µg / mL each. Cells were incubated for one hour at 4°C, rinsed three times with PBS, fixed with 4% paraformaldehyde, and blocked (Avidin / Biotin Blocking Kit, SP-2001, Vector Laboratories) prior to applying Anti-AAV9 (1:200 dilution, ThermoFisher, 7103332100) to the wells treated with BI-hTFR1 or the wells with no virus, or Anti-AAVX (1:200 dilution, ThermoFisher, 7103522100) to the wells treated with AAV2. After three PBS washes to clear away unbound primary antibodies, Streptavidin 555 (1:500 dilution, Thermofisher, S21381) and Alexa Fluor© 488 (1:500 dilution, JacksonImmunoResearch, 115- 545-003) were applied for the visualization of AAVs and TfR1, respectively.

[0135] To image AAV colocalization with Tf-647, BI-hTFR1 or AAV2 were added to hCMEC / D3 cells in optical plates at 25,000 vg / cell with Tf-647 (1 µg / mL). Cells were incubated for one hour at 4°C, followed by immunostaining as described above.

[0136] To image AAV and TfR1 colocalization, BI-hTFR1 or AAV2 were added to hCMEC / D3 cells in optical plates at 25,000 vg / cell. Cells were incubated for one hour at 37°C, prepared for immunostaining as described above but with the addition of a permeabilization step prior to the blocking of endogenous biotin to visualize TfR1. Streptavidin 555 (1:500 dilution, Thermofisher, S21381) and goat anti-mouse 647 nm (1:500 dilution, 115-605-062, Jackson ImmunoResearch) were applied.

[0137] For organelle colocalization, the following conjugated reagents were applied: Rab5 ((D-11) FITC, 1:100 dilution, Santa Cruz Biotechnology, sc-46692), Rab7 (1:400 dilution, abcam, ab198337), KDEL (1:500 dilution, abcam, ab184819), RCAS1 (1:200 dilution, Cell Signaling Technology, 12290), and TGN46 (1:50 dilution, abcam, ab50595). For unconjugated antibodies (RCAS1 and TGN46), goat anti-rabbit 488 (Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L), 1:500 dilution, JacksonImmuno, 111-545-144) was applied to visualize the staining. For wells requiring dual mouse antibodies (those co-stained with Rab5 and OKT9), cells were blocked for one hour with mouse-on-mouse blocking reagent (ReadyProbes™™ Mouse-on-Mouse IgG Blocking Reagent, 1:30 dilution, ThermoFisher, R37621) between antibody staining procedures.

[0138] Images for all experiments were obtained with a 60X oil objective on a Nikon Ti-e inverted spinning disc confocal microscope, with the exception of TGN46 staining, which was performed using a 63X oil objective on a Zeiss LSM 900 confocal microscope. Within each experiment, images were taken with predetermined, optimized, and fixed exposure times to allow image comparisons.Imaging and tissue cell counting

[0139] Images of native mScarlet in the whole brain, cortex, striatum, thalamus, spinal cord, liver, or the dorsal root ganglion were taken on a Keyence BZ-X810. For images of whole brain sections, single plane tiled images were taken with autofocus mode on and stitched using the Keyence Analysis Software. For comparisons of native mScarlet fluorescence across groups, imaging settings were optimized for BI-hTFR1 in TFRC KI mice and then applied across all samples and all groups within each organ. Adjustments to the levels and gamma in the whole brain, cortex, striatum, thalamus, spinal cord, liver, and dorsal root ganglion images were performed using identical settings across all samples within each brain region or tissue type.

[0140] Stained sections were imaged on a Keyence BZ-X810 using sectioning mode with a Z- stack depth of 10 µm during acquisition. For cell counting, image settings were exposed to detect low mScarlet expressing cells in each sample group within each brain region. The Z-stack was reconstructed into a full-single-stack image using Keyence Analysis Software and exported as separate TIFs for each channel imaged. Images were then imported as input data into ilastik version 1.4.0 (66) where the Pixel Classification within the segmentation workflow was used. All color / intensity, edge, and texture features were selected and used during training. Training was performed to distinguish foreground and background pixels across images that represented the cortex, striatum, or thalamus in C57BL / 6J or TFRC KI mice that had received BI-hTFR1 or AAV9 in NeuN or SOX9 stained brain sections. Predictions were then exported as probabilities in TIF format and then imported into CellProfiler 4.2.4 (67). A custom workflow was created to determine percent overlap between positively transduced cells and antibody markers. First, channels were aligned using the Align module. Second, the IdentifyPrimaryObjects module with Otsu two classes thresholding method was used to detect and segment foreground objects (positively transduced cells, NeuN-positive, or SOX9-positive cells) from background in the images. Third, foreground objects were then expanded by 2-5 pixels using the ExpandOrShrinkObjects module. Fourth, the MaskObjects module was used to determine the number of colocalized objects that overlap with a threshold of 40% overlap or more. A separate CellProfiler workflow was created where the Identify Primary Objects module was used to detect foreground and background objects (two class) and then the OverlayOutlines module was used to overlay an outline of detected foreground objects onto the raw input image of the mScarlet, NeuN, or SOX9 channel. The images were exported with the SaveImages module. The exported image was then used to verify if further training with the Pixel Classification in ilastik was needed in order to detect missed or improperly classified cells. An independent evaluator without prior knowledge of expected outcomes conducted a manual count of randomly selected images using the Photoshop Count Tool to determine the percent overlap of mScarlet-positive cells with NeuN or SOX9-positive cells in stained tissue. The results were evaluated to be within ± 5% deviation.Western blot

[0141] Purified N-terminal flag tagged full length human and mouse TfR1 proteins or whole mouse brain lysates were separated on Bolt 4-12% Bis-Tris Plus gels and transferred onto nitrocellulose membranes. After incubation with anti-TfR1 antibodies (0.5 µg / mL ThermoFisher, Catalog # 13-6890, 0.25 µg / mL R&D, AF2474) or anti-ẞ-actin (1:5000; Abcam, AB20272) followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody. The detection of the HRP signal was by SuperSignal West Femto Maximum Sensitivity Substrate using a Bio-Rad ChemiDoc TM MP system #1708280.In vivo biodistribution, transduction, and Luciferase assays

[0142] BI-hTFR1 or AAV9 encoding CAG-NLS-mScarlet-P2A-Luciferase-WPRE-SV40pA were administered intravenously to 18 to 21-week-old female C57BL / 6J or TFRC KI mice at a dose of 5 x 1011 vg / mouse. After three weeks, the mice were perfused with PBS. Tissue samples from the brain, liver, spinal cord, and DRGs were dropped fixed into 4% PFA. Remaining tissues, including additional samples of the brain, liver, spinal cord, and DRGs were collected snap frozen on dry ice and stored at -80°C. Biodistribution analysis: Samples were processed using a DNeasy 96 Blood & Tissue Kit (Qiagen, 69581). qPCR was performed for the mScarlet transgene and the mouse glucagon gene as previously described (see primer sequences in table S1) (5). For mRNA analysis, RNA was isolated using TRIzol (Invitrogen, 15596026) and purified using an RNeasy 96 Kit (Qiagen, 74171). cDNA synthesis was performed using Maxima H Minus Reverse Transcriptase (Thermo Scientific, EP0753). qPCR for mScarlet and mouse GAPDH was performed (primer sequences in table S1). Luciferase assays: Protein was extracted from tissue samples using the lysis buffer T-Per with 1X Halt protease inhibitor (Invitrogen, 78430) while kept ice cold at all times. 10 µg of total protein was used to assess the reporter gene using the Britelite Plus reporter gene assay system.GBA1 in vivo delivery experiment

[0143] BI-hTFR1 or AAV9 encoding CMV / CBA-GBA1-HA-pA was intravenously injected into 28 to 30-week-old female TFRC KI mice at a dose of 1 x 1014 or 5 x 1012 vg / kg. After three weeks, cerebrospinal fluid (CSF) was collected from the cisterna magna of anesthetized mice as previously described (68). Briefly, the cisterna magna was surgically exposed and punctured with a sharpened glass capillary, and CSF was allowed to flow into the capillary. Approximately 6-10 µL of CSF was collected from each animal. Then, the thoracic cavity was exposed and 200-400 µL of blood was collected into a microtainer tube with serum separator additive (BD, 365967). The mice were then transcardially perfused with ice cold PBS. Brain, liver, and spinal cord tissue sections were drop fixed into 4% PFA. Additional tissue samples from these organs were collected and snap frozen at -80°C. After a 1.5-hour incubation at room temperature, the blood was centrifuged at 10,000 ×g for 90 seconds, and serum was collected and stored at -80°C. IHC was performed on fixed tissues as described in the Animals section of the Methods. GBA-HA was detected with an anti-HA primary antibody (Sigma, 11867423001) at a 1:50 dilution, followed by an AlexaFluor568-conjugated secondary antibody at a 1:500 dilution (Invitrogen, A78946). Vector genomes were quantified using qPCR primers targeting CMV and mouse GAPDH (table S1).

[0144] To measure GCase activity, whole tissue lysates were prepared. Tissue was thawed on ice, weighed, and homogenized in a 2x volume of ultra-pure distilled water with complete protease inhibitor (Sigma, 4693132001) using the GenoGrinder (SPEX Sample prep). Homogenate was then centrifuged at 10,000 ×g for 2 minutes, transferred to a new, pre-chilled 1.5 mL Eppendorf tube, and incubated on ice for 15-30 minutes. Next, the samples were centrifuged at 12,000 ×g for 20 minutes. Supernatant was collected and stored at -80°C or used in subsequent analysis. A BCA assay (Thermo Fisher Scientific, 23225) was used to determine total protein concentration in the resulting lysates, as described by the product manufacturer. The SensoLyte Red Glucocerebrosidase (GBA) Activity Assay Kit (AnaSpec, AS-72259) was used to determine enzyme activity in serum and whole tissue lysate samples. Briefly, 20 µL of serum or the volume of tissue lysate containing 30 µg of protein was added into each well. Assay buffer was added to a total volume of 50 µL. Then, 50 µL of 1X enzyme substrate was added to each well. Samples were gently mixed and incubated for one hour at room temperature. The fluorescence signal at an excitation / emission of 570 / 610 nm was measured in end-point mode using the EnVision 2104 plate reader (PerkinElmer). A resorufin reference fluorescence standard curve was used to determine the amount of reaction product in each well from the measured relative fluorescence units (RFU). The resulting values were used to calculate enzyme activity, expressed in µU per mL of blood or µU per mg of protein. One unit of GCase activity is the amount of enzyme that generates 1 µmol of product per minute under the specified reaction conditions.Table $1. Primers and barcodes used for qPCR in this study. Each sequence is listed 5' to 3'.Primer namePrimer sequencehTfR1-qPCR-FGCTATGGGACTATTGCTGTGATCGTC (SEQ ID NO: 43844)hTfR1-qPCR-RCCAATATAAGCGACGTGCTGCAG (SEQ ID NO: 43845)msTfR1-qPCR-FTGTGGAGATGAAACTGGCTGC (SEQ ID NO: 43846)msTfR1-qPCR-RAGTGCAATAGCTGCAAAGCAGAG (SEQ ID NO: 43847)marTfR1-qPCR-FAACAAGAGCGTGAAGCTCTCTG (SEQ ID NO: 43848)marTfR1-qPCR-RCTCCAGGACCCCAGGAGTC (SEQ ID NO: 43849)macTfR1-qPCR-FGGAAAACAAGAGTGTGAAGCTCACG (SEQ ID NO: 43850)macTfR1-qPCR-RGGAGAGCTGTCCCCACACTG (SEQ ID NO: 43851)MSCARLET-QPCR-FCCGTGACCCAGGACACCTC (SEQ ID NO: 43852)mScarlet-qPCR-R-biodCTTCTTCTGCATTACGGGGC (SEQ ID NO: 43853)mScarlet-qPCR-R-bindingGCCATCTTAATGTCGCCCTTCAG (SEQ ID NO: 43854)CMV-qPCR-FTGTTCCCATAGTAACGCCAATAG (SEQ ID NO: 43855)CMV-qPCR-RGTACTTGGCATATGATACACTTGATG (SEQ ID NO: 43856)GAPDH-qPCR-FACCACAGTCCATGCCATCAC (SEQ ID NO: 43857)GAPDH-qPCR-RTCCACCACCCTGTTGCTGTA (SEQ ID NO: 43858)mGAPDH-qPCR-FCGCCCTGATCTGAGGTTAAAT (SEQ ID NO: 43859)mGAPDH-qPCR-RCGGAGCAACAGATGTGTGTA (SEQ ID NO: 43860)mGlucagon-qPCR-FAAGGGACCTTTACCAGTGATGTG (SEQ ID NO: 43861)mGlucagon-qPCR-RАСТТАСТCTCGCCTTCCTCGG (SEQ ID NO: 43862)msTfR1-exonland2-FCTAACTTGTTTGGTGGGGAAC (SEQ ID NO: 43863)msTfR1-cxonland2-RCTTCATCTGCAGCCAGTTTCAT (SEQ ID NO: 43864)genotyping-h&mTFRC-FCTCGTGAGGCTGGATCTCAAAAAGATG (SEQ ID NO: 43865)genotyping-hTFRC-RCACCAGGTAAACAAGTCTACCGTTCTTATC (SEQ ID NO: 43866)genotyping-mTfrc-RACTGGGTCTAAGTTACCATTTGACTGC(SEQ ID NO: 43867)BI-hTFR1_barcode_qPCR_FTCGAGACCATAGATGACTACCC (SEQ ID NO: 43868)BI-hTFR1_barcode_qPCR_RGATCTGTGTTGTTGCGGTATAATG (SEQ ID NO: 43869)AAV2_barcode_qPCR_FTCGAGATGGAGGAAGGAACT (SEQ ID NO: 43870)AAV2_barcode_qPCR_RAGATCTGCTGTAACCCACAAA (SEQ ID NO: 43871)AAV9_barcode_qPCR_FTCGAGCATGCGGGTAGAT (SEQ ID NO: 43872)AAV9_barcode_qPCR_RCTGTCTTGGATGTGATTGCTGTA (SEQ ID NO: 43873)BI-hTFR1 genome barcodeCTCGAGACCATAGATGACTACCCATCCCATCGAGCGTTGT ACCCTATTCAGAGGCAGACATTATACCGCAACAACACAGA TCT (SEQ ID NO: 43874)AAV2 genome barcodeCTCGAGATGGAGGAAGGAACTTTTGCGTGTGAGCGTTGTA CCCTATTCAGAGTGACACGTTTGTGGGTTACAGCAGATCT (SEQ ID NO: 43875)AAV9 genome barcodeCTCGAGCATGCGGGTAGATCATGTGGGTAGGAGCGTTGTA CCCTATTCAGAGGGGTTACAGCAATCACATCCAAGACAGA TCT (SEQ ID NO: 43876)Table S2. Production and purification methods used in this study with final post-purification yields.Study GenomeProduction Purification ddPCR target AAV9BI-hTFR1 Unit1 CAG-NLS-GFP-P2A-Luc adherent static affinity CAG4.10E+112.06E+11 vg / 150mmHEK293T2 CAG-NLS-GFP-P2A-Luc suspension iodixanol WPRE2.78E+13 9.93E+12 vg / LHEK293T3 CMV-CBA-hGBA-HAsuspension iodixanolCAG1.74E+13 1.25E+13 vg / LHEK293TTable S3. Human GBA1 NT with HA tag (underlined and bolded) sequence used in the vectoratggagttttcaagtccttccagagaggaatgtcccaagcctttgagtagggtaagcatcatggctggcagcctcacaggattgcttctacttcaggcagtgtc gtgggcatcaggtgcccgcccctgcatccctaaaagcttcggctacagctcggtggtgtgtgtctgcaatgccacatactgtgactcctttgaccccccgacct ttcctgcccttggtaccttcagccgctatgagagtacacgcagtgggcgacggatggagctgagtatggggcccatccaggctaatcacacgggcacagge ctgctactgaccctgcagccagaacagaagttccagaaagtgaagggatttggaggggccatgacagatgctgctgctctcaacatccttgccctgtcaccc cctgcccaaaatttgctacttaaatcgtacttctctgaagaaggaatcggatataacatcatccgggtacccatggccagctgtgacttctccatccgcacctac acctatgcagacacccctgatgatttccagttgcacaacttcagcctcccagaggaagataccaagctcaagatacccctgattcaccgagccctgcagttgg cccagcgtcccgtttcactccttgccagcccctggacatcacccacttggctcaagaccaatggagcggtgaatgggaaggggtcactcaagggacagcc cggagacatctaccaccagacctgggccagatactttgtgaagttcctggatgcctatgctgagcacaagttacagttctgggcagtgacagctgaaaatgag ccttctgctgggctgttgagtggataccccttccagtgcctgggcttcacccctgaacatcagegagacttcattgcccgtgacctaggtcctaccctcgccaa cagtactcaccacaatgtccgcctactcatgctggatgaccaacgcttgctgctgccccactgggcaaaggtggtactgacagacccagaagcagctaaata tgttcatggcattgctgtacattggtacctggactttctggctccagccaaagccaccctaggggagacacaccgcctgttccccaacaccatgctctttgcctc agaggcctgtgtgggctccaagttctgggagcagagtgtgcggctaggctcctgggatcgagggatgcagtacagccacagcatcatcacgaacctcctgt accatgtggtcggctggaccgctggaaccttgccctgaaccccgaaggaggacccaattgggtgcgtaactttgtcgacagtcccatcattgtagacatca ccaaggacacgttttacaaacagcccatgttctaccaccttggccacttcagcaagttcattcctgagggctcccagagagtggggctggttgccagtcagaa gaacgacctggacgcagtggcactgatgcatcccgatggctctgctgttgtggtcgtgctaaaccgctcctctaaggatgtgcctcttaccatcaaggatcctg ctgtgggcttcctggagacaatctcacctggctactccattcacacctacctgtggcgtcgccagtatccatatgacgtcccagactatgca (SEQ ID NO: 43877)Table S4. hGBA1-HA AA Sequence used in the vector:MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDP PTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNIL ALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHR ALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQF WAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWA KVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGS WDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLG HFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGY SIHTYLWRRQYPYDVPDYA (SEQ ID NO: 43878)***

[0145] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

Claims

What is claimed is:

1. An engineered recombinant adeno associated virus (AAV) particle comprising an engineered AAV capsid polypeptide comprising a transferrin receptor (TfR1) binding moiety and a recombinant AAV genome comprising a polynucleotide encoding GBA1 operably linked to a regulatory element that promotes expression of GBA1 in the central nervous system (CNS) flanked by AAV ITR sequences.

2. The engineered recombinant AAV particle of claim 1, wherein the TfR1 binding moiety is inserted or substituted in loop IV, loop VIII, or both of an AAV capsid protein.

3. The engineered recombinant AAV particle of any of the preceding claims, wherein the AAV capsid polypeptide is VP1, VP2, VP3, or a combination thereof.

4. The engineered recombinant AAV particle of any of the preceding claims, wherein the TfR1 binding moiety is inserted between amino acids 588 and 589 of a capsid protein of AAV9, or in an analogous position of a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10.

5. The engineered recombinant AAV particle of any of the preceding claims, wherein the TfR1 binding moiety is inserted between two consecutive amino acids within amino acid 451-460 of a capsid protein of AAV9, or in an analogous position of a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh. 10.

6. The engineered recombinant AAV particle of any of the preceding claims, wherein the TfR1 binding moiety comprises of a 7-mer, the 7-mer comprises of LHRLGPN (SEQ ID NO: 36834), YSRIGPN (SEQ ID NO: 42487), LHRLGPD (SEQ ID NO: 36413), LHRAGPD (SEQ ID NO: 36894), YSRIGPD (SEQ ID NO: 38223), LSRIGPD (SEQ ID NO: 36274), LARSGPD (SEQ ID NO: 18035), YSRNSDN (SEQ ID NO: 42488), LHKAGPN (SEQ ID NO: 36305), LSRIGPN (SEQ ID NO: 36347), LAKSGPN (SEQ ID NO: 36287), YARNGPN (SEQ ID NO: 14048), FRSTNGV (SEQ ID NO: 16070), VESTNGR (SEQ ID NO: 36431), VDSTNGV (SEQ ID NO: 12206), VQSTNGV (SEQ ID NO: 36423), VSSTNGV (SEQ ID NO: 12333), TESTNGR (SEQ ID NO: 17558), VQSTNGI (SEQ ID NO: 11292), FVSTNGV (SEQ ID NO: 42491), RGEDVYP (SEQ ID NO: 36864), RLEDVFP (SEQ ID NO: 36264), RTYDSYP (SEQ ID NO: 37938), RTYDAYP (SEQ ID NO: 38571), RTYDSFP (SEQ ID NO: 37806), RTETVYP (SEQ ID NO: 36486), RTETVFP (SEQ ID NO: 36389), RTEHVFP (SEQ ID NO: 36603), LCKPCLD (SEQ ID NO: 36437), LCKPCPT (SEQ ID NO: 36438), KDEFTTF (SEQ ID NO: 36308), KDDFTTY (SEQ ID NO: 36336), RDEFTTY (SEQ ID NO: 36615), KDEFSTY (SEQ ID NO: 36390), RDEFTSF (SEQ ID NO: 36701), REDHVSW (SEQ ID NO: 37067), IALKGWD (SEQ ID NO: 36248), NALEGRD (SEQ ID NO: 36407), VALEGRD (SEQ ID NO: 36604), VALKGWD (SEQ ID NO: 17701), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), YHRLSNN (SEQ ID NO: 16636), VHRLQDK (SEQ ID NO: 16602), LHALSHN (SEQ ID NO: 16608), PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16199), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 16206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGI (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSNGV (SEQ ID NO: 16123).

7. The engineered recombinant AAV particle of claim 6, wherein the 7-mer comprises YSRIGPN (SEQ ID NO: 42487).

8. The engineered recombinant AAV particle of any one of claims 1 to 5, wherein the TfR1 binding moiety is selected from any of SEQ ID NO: 42513-43842.

9. A pharmaceutical composition comprising the engineered recombinant AAV particle of any one of claims 1 to 8 and an acceptable carrier.

10. A method of delivering GBA1 to the central nervous system (CNS) comprising administering the pharmaceutical composition of claim 9.

11. The method of claim 10, wherein the pharmaceutical composition is administered at a dosage of between 0.1 x 1012 vg / kg to 1 x 1014 vg / kg.

12. The method of claim 11, wherein the pharmaceutical composition is administered at a dosage of 0.1 x 1012 vg / kg to 100 x 1012 vg / kg.

13. The method of claim 12, wherein the pharmaceutical composition is administered at a dosage of 1 x 1012 vg / kg to 10 x 1012 vg / kg.

14. The method of claim 13, wherein the pharmaceutical composition is administered at a dosage of 5 x 1012 vg / kg.

15. The method of any of claims 10 to 14, wherein the subject suffers from Gaucher's disease.

16. The method of any of claims 10 to 14, wherein the subject suffers from Parkinson's disease.

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