Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Jul 22.
Published in final edited form as: Science. 2024 May 16;384(6701):1220–1227. doi: 10.1126/science.adm8386

An AAV capsid reprogrammed to bind human transferrin receptor mediates brain-wide gene delivery

Qin Huang 1,†, Ken Y Chan 1,†, Jason Wu 1, Nuria R Botticello-Romero 1, Qingxia Zheng 1, Shan Lou 1, Casey Keyes 1, Alexander Svanbergsson 1, Jencilin Johnston 1, Allan Mills 1, Chin-Yen Lin 1, Pamela P Brauer 1, Gabrielle Clouse 1, Simon Pacouret 1, John W Harvey 1, Thomas Beddow 1, Jenna K Hurley 1, Isabelle G Tobey 1, Megan Powell 1, Albert T Chen 1, Andrew J Barry 1, Fatma-Elzahraa Eid 1,2, Yujia A Chan 1, Benjamin E Deverman 1,*
PMCID: PMC13387621  NIHMSID: NIHMS2121597  PMID: 38753766

Abstract

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an adeno-associated virus (AAV) capsid, BI-hTFR1, that binds human transferrin receptor (TfR1), a protein expressed on the blood-brain barrier (BBB). BI-hTFR1 was actively transported across human brain endothelial cells 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. These findings establish BI-hTFR1 as a potential vector for human CNS gene therapy.

One sentence summary:

A candidate AAV vector for CNS gene therapy crosses the blood-brain barrier by binding human transferrin receptor.

A critical challenge in the development of efficient gene therapy vectors is engineering delivery vehicles with mechanisms of action (MOAs) applicable to human patients. Conventional approaches to engineering vectors such as adeno-associated viruses (AAVs) largely rely on capsid selections in animals. These selections have been used extensively because they do not require prior knowledge of an MOA (1–4). However, capsids selected in animals mostly have not translated across preclinical models, and AAVs with clear translational potential and known MOAs for entering the central nervous system (CNS) have not been described. Here, we 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 one capsid crossed the blood-brain barrier (BBB) and mediated efficient CNS-wide gene delivery.

We targeted TfR1 for its high expression on the human BBB; ability to mediate ligand-independent receptor-mediated transcytosis (RMT) across the CNS vasculature (5–9); and track record as a target to shuttle biologics into the CNS in animals (10–14) and humans as investigational therapies (15–18) and an approved therapeutic for mucopolysaccharidosis type II (19). We screened 7-mer-modified AAV9 capsid libraries for their ability to bind to human TfR1 in vitro. The top-performing capsid, AAV-BI-hTFR1, exhibited more efficient gene delivery to and improved active transport across human brain endothelial cells. When systemically administered to adult human TFRC knock-in (KI) mice, BI-hTFR1 transduced most neurons and astrocytes across multiple brain regions. The enhanced tropism depended on an interaction with the humanized TfR1 and was CNS-specific, consistent with the high level of TFRC expression on the CNS vasculature relative to other organs (9).

To explore its ability to deliver a therapeutically relevant cargo, we intravenously injected TFRC KI mice with BI-hTFR1 or AAV9 packaging the human glucosylceramidase beta 1 (GBA1) gene. Inactivating mutations of GBA1 cause Gaucher’s disease, a lysosomal storage disorder often affecting the CNS, and GBA1 mutations are a risk factor for Parkinson’s disease and dementia with Lewy Bodies (20). Systemic delivery of BI-hTFR1:GBA1 in adult TFRC KI mice achieved robust brain-wide expression and elevated glucocerebrosidase (GCase) activity in the brain and cerebrospinal fluid (CSF), which were not observed with AAV9. BI-hTFR1 represents a promising vector for the development of CNS-targeting human gene therapies.

Results

AAV capsids engineered to bind human TfR1

We screened AAV9-based capsid libraries with randomized 7-mer insertions between viral capsid protein (VP1) residues 588–589 for selective binding to human TfR1 using our recently described receptor-targeting approach (21) (Fig. 1A), and chose four capsids with a common sequence motif for validation: BI-hTFR1, BI-hTFR1–2, BI-hTFR1–3, and BI-hTFR1–4. We used each capsid and AAV9 (subsequent references to AAV9 refer to the unmodified AAV) to package a single-stranded (ss) genome expressing nuclear mScarlet and luciferase driven by the ubiquitous early cytomegalovirus (CMV) enhancer/chicken β actin (CAG) promoter. Relative to AAV9, the TfR1-binding capsids exhibited greater association with and transduction of Chinese hamster ovary (CHO) cells stably expressing human TFRC but not the control CHO cells or those expressing TFRC from rhesus macaque, marmoset, or mouse (Fig. 1B and C, and fig. S1A–D). We tested their ability to associate with and transduce human brain endothelial cells that endogenously express TFRC. Although the amounts of AAV associated with primary human brain microvascular endothelial cells (hBMVECs) and a human brain endothelial cell line (hCMEC/D3) varied (Fig. 1D and fig. S1E), each TfR1-binding capsid exhibited enhanced transduction of both cell types compared to AAV9 (Fig. 1E and fig. S1F). To assess the relative binding affinities of the capsids with purified human TfR1, we performed bio-layer interferometry (BLI). AAV9 showed no detectable TfR1 binding while each BI-hTFR1 capsid showed concentration-dependent binding (Fig. 1F). We derived kinetic constants using a multi-phasic binding model (fig. S2A) based on rationale provided in the Materials and Methods (22). Based on its cell assay performance and ability to bind human TfR1 at lower concentrations, we chose BI-hTFR1 for further investigation.

Fig. 1. AAV9 can be programmed to bind human TfR1.

Fig. 1.

Open in a new tab

(A) An AAV9 (K449R)-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 an ssAAV-CAG-NLS-mScarlet-P2A-Luciferase-WPRE-SV40pA genome exhibited enhanced species-specific (B) association with and (C) transduction (luciferase activity, normalized relative light units [RLU]) of CHO cells stably expressing TFRC (two-way ANOVA using AAV9 as the main comparison group for each cell line with 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 (D) association with and (E) transduction (luciferase activity) of hBMVEC and hCMEC/D3 cells (one-way ANOVA using AAV9 as the comparison group for each cell line with Dunnett’s multiple comparison correction: ****, ***, and * indicate p ≤ 0.0001, ≤ 0.001, and ≤ 0.05, respectively; n = 4 replicates, error bars indicate ± SEM). Values in (B–E) are normalized to AAV9 (reported as fold change) in each cell line. (F) 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. 2:1 binding model curve fits (black lines) are shown.

BI-hTFR1 binds the TfR1 apical domain

To evaluate whether TfR1 binding is necessary for the increased transduction of brain endothelial cells by BI-hTFR1, we assessed hCMEC/D3 transduction in the presence of the OKT9 antibody that binds the TfR1 apical domain (23) or the AF2474 antibody that competes with transferrin (Tf) for TfR1 binding. We observed transduction inhibition by increasing concentrations of OKT9 but not AF2474 (Fig. 2A). Consistent with this result, OKT9 directly competed with BI-hTFR1 for binding to purified human TfR1 (fig. S2B). Binding to the TfR1 apical domain is a common feature of several antibody-based BBB shuttles that have been tested in humans. Binding a site that does not overlap with Tf binding is important because surface TfR1 is thought to be mostly occupied by Tf based on the typical 10–20 μM blood concentration of iron-bound holo-Tf (24) and its sub nanomolar TfR1 binding affinity (fig. S2C). We found that BI-hTFR1 binds human TfR1 with similar kinetics in the presence or absence of a receptor-saturating concentration of holo-Tf (Fig. 2B and fig. S2D) and that BI-hTFR1 can bind a preformed complex of TfR1 and Tf (Fig. 2C). In addition, OKT9, but not Tf, inhibited BI-hTFR1 association with hCMEC/D3 cells and colocalization with TfR1 (Fig. 2D and fig. S3).

Fig. 2. Human TfR1-targeted capsids transduce human brain endothelial cells via interactions with the apical domain of human TfR1.

Fig. 2.

Open in a new tab

(A) Transduction (luciferase activity, normalized RLU) of hCMEC/D3 cells incubated with 3 × 108 vector genomes (vg)/mL of the indicated AAV and specified concentrations of OKT9 or AF2474 antibody is shown (two-way ANOVA using the no antibody control for each condition as the main comparison group with Bonferroni multiple comparison correction: **** indicates p ≤ 0.0001; n = 3 replicates, error bars indicate ± SEM). (B) 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 had or had not been pre-incubated with 300 nM holo-Tf was used as an analyte. 2:1 binding model curve fits (black lines) are shown. (C) Biotinylated holo-Tf was immobilized on streptavidin-coated BLI probes, introduced first into human TfR1, then into buffer with or without OKT9, and finally into BI-hTFR1. Gray segments highlight each indicated association step. (D) 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. S3).

Active trafficking of BI-hTFR1 across a human BBB model

To test AAV transport across a human endothelial cell barrier, we established a BBB transwell model using hCMEC/D3 cells and compared BI-hTFR1, AAV9, or AAV2 transport across the barrier. To minimize the effect of well-to-well variation, we mixed BI-hTFR1, AAV9, and AAV2 carrying individually identifiable barcoded genomes, added this to the media in the upper chambers, maintained at 37°C or 4°C, and measured transport to the lower chambers (Fig. 3A). At 4°C, transcytosis is suppressed but passive transport can occur, providing a measurement of leakiness (25). As previously reported, AAV9 crossed the barrier more efficiently than AAV2 at 37°C, and the number of vector genomes (vg) detected in the bottom chamber was reduced at 4°C for both AAVs (26–28). Compared to AAV9 and AAV2, more BI-hTFR1 was actively transported across the barrier (Fig. 3B).

Fig. 3. BI-hTFR1 is efficiently endocytosed and actively transported across human brain endothelial cells.

Fig. 3.

Open in a new tab

(A) The transwell BBB model experimental design is shown. (B) 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). (C) 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.

RMT involves endocytosis, vesicular trafficking, and exocytosis. We explored whether BI-hTFR1 transcytoses hCMEC/D3 cells via the TfR1 RMT pathway by assessing its colocalization with various organelle markers. We found that 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. S4). In contrast, AAV2 was not highly colocalized with any of the markers except TGN46; colocalization with TGN46 is consistent with the requirement for AAV trafficking to the trans-Golgi for transduction (29). Cell surface TfR1 clustering promotes clathrin-coated pit formation and uptake into a subpopulation of endosomes enriched in RAB5 (30). A smaller proportion of TfR1 colocalizes with RAB7 (31), which decorates endosomes associated with transcytosis and the lysosomal degradation pathway. These findings are consistent with our observation that BI-hTFR1 colocalized with RAB5 and RAB7 to a greater degree than AAV2.

CNS tropism of BI-hTFR1

We next investigated whether BI-hTFR1 could cross the BBB in vivo by engaging human TfR1. We used TFRC 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). The mouse-human hybrid mRNA and protein levels were similar to those in C57BL/6J wild type (WT) mice (fig. S5). We intravenously injected BI-hTFR1 or AAV9 into adult female WT or TFRC KI mice. Three weeks post-injection, we observed enhanced biodistribution to and transduction of the brain and spinal cord by BI-hTFR1 in TFRC KI but not WT mice (Fig. 4 and fig. S6A). Increased biodistribution and transduction relative to AAV9 were not observed in other organs assessed, indicating that the interaction with human TfR1 selectively enhanced CNS-targeting. Comparing the TFRC KI mice that received BI-hTFR1 to the WT mice that received AAV9, we detected 6-fold and 12-fold more viral genomes, 54-fold and 43-fold higher AAV mRNA transcript levels, and 132-fold and 58-fold greater luciferase activity in the brain and spinal cord, respectively. AAV9 transduction of the CNS was not affected by genotype. Consistent with these data, mScarlet levels in the livers of WT and TFRC KI mice were comparable between AAV9 and BI-hTFR1 (fig. S6B and C). We observed reduced BI-hTFR1 biodistribution to the dorsal root ganglion (DRG) and spleen in WT and TFRC KI mice relative to AAV9 (Fig. 4B, DRG transduction is shown in fig. S6D), suggesting that this is due to a TfR1-binding-independent change introduced by the 7-mer insertion in BI-hTFR1.

Fig. 4. BI-hTFR1 efficiently delivers genes to the CNS of TFRC KI mice and transduces neurons and astrocytes throughout.

Fig. 4.

Open in a new tab

(A) In TFRC KI mice, mouse Tfrc exons 4–19 encoding the extracellular region of TfR1 have been replaced by those of human TFRC. BI-hTFR1 or AAV9 carrying an ssAAV-CAG-NLS-mScarlet-P2A-Luciferase-WPRE-SV40pA genome were intravenously injected into adult female C57BL/6J or TFRC KI mice at 5 × 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 (B) biodistribution reported as vector genomes per mouse genome and (C) luciferase activity within different organs are shown at three weeks post-injection (two-way ANOVA using BI-hTFR1 in TFRC KI mice as the main comparison group with 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). Representative (D) whole brain and (E) spinal cord images from each group of mice at three weeks post-injection are shown. Scale bars (D) = 1 mm, (E) = 100 μm. The percentage of (F) NeuN, (G) SOX9, (H) OLIG2, and (I) ERG cells positive for the indicated marker that expressed mScarlet in the cortex, striatum, and thalamus are shown (two-way ANOVA was used with BI-hTFR1 in TFRC KI mice as the main comparison group with Bonferroni multiple comparison correction: **** and *** indicate p ≤ 0.0001 and ≤ 0.001, respectively; each data point represents an individual mouse, error bars indicate ± SEM).

To evaluate the transduction of specific CNS cell types, we assessed the fraction of mScarlet-expressing neurons, astrocytes, oligodendrocyte lineage cells, or endothelial cells identified by NeuN, SOX9, OLIG2, or ERG staining, respectively. In TFRC KI mice, we observed efficient BI-hTFR1 transduction of NeuN+, SOX9+, and OLIG2+ cells: a mean of 54, 32, and 71% of NeuN+ cells and a mean of 86, 84, and 92% of SOX9+ cells in the cortex, striatum, and thalamus, respectively (Fig. 4F and G, and fig. S7A and B) and a mean of 34%, 19%, and 31% of OLIG2+ cells in the cortex, striatum, and corpus callosum, respectively (Fig. 4H and fig. S7C). In the WT mice injected with BI-hTFR1 or either line injected with AAV9, we detected less than 6%, 20%, and 3% transduction of NeuN+, SOX9+, and OLIG2+cells, respectively, in these brain regions. We quantified similar percentages (37–47%) of ERG+ cells in the cortex, striatum, and thalamus transduced by BI-hTFR1 in TFRC KI mice or AAV9 in either line; BI-hTFR1 was less efficient at transducing ERG+ cells in WT mice (Fig. 4I and fig. S7D).

BI-hTFR1 mediates brain-wide GBA expression

We next evaluated BI-hTFR1’s ability to deliver human GBA1 to TFRC KI mice. We packaged an ssAAV containing a cytomegalovirus enhancer-chicken beta-actin promoter (CMV-CBA) driving the expression of human GBA1 with an influenza virus hemagglutinin (HA) tag into AAV9 or BI-hTFR1 (Fig. 5A). The AAVs were intravenously administered to TFRC KI mice at a dose of 1 × 1014 vg/kg, comparable to that of the AAV9-based Zolgensma gene therapy for spinal muscular atrophy (32). Additionally, we administered BI-hTFR1:GBA1 to a separate group of TFRC KI mice at a 20-fold lower dose. For the untreated control, we used C57BL/6J mice. Three weeks post-administration, we detected ~30 times more AAV genomes in the brains of BI-hTFR1-treated mice compared to AAV9-treated mice (Fig. 5B). We observed brain-wide expression of GBA-HA (Fig. 5C, and fig. S8A and B) and elevated GCase activity in brain tissue lysates and the CSF of BI-hTFR1-treated mice, compared to AAV9-treated or untreated mice (Fig. 5D); brain GCase activity mediated by BI-hTFR1 and AAV9 was 6.7-fold and 1.1-fold, respectively, that of the untreated mice. We detected GBA-HA primarily in neurons and astrocytes but minimally in the endothelium (fig. S9A). In AAV9-treated mice, we did not detect increased brain GBA expression but detected similar amounts of AAV genomes, GBA-HA, and GCase activity in the liver compared to BI-hTFR1-treated mice (Fig. 5B and D, and fig. S8C). Reduced GCase activity in the brain and CSF has been associated with Parkinson’s disease (33), and studies suggest that GBA1 delivery to the brain can treat neuronal α-synucleinopathy (34). Notably, in mice that received the low dose of BI-hTFR1, we observed neuronal transduction in regions linked to motor symptoms of Parkinson’s disease such as the substantia nigra pars compacta, deep cerebellar nuclei, and brainstem (Fig. 5C and fig. S8A) (35). In contrast, we did not detect GBA-HA in the brains of mice treated with the 20-fold higher dose of AAV9.

Fig. 5. BI-hTFR1 efficiently delivered GBA1 and increased GCase activity in the brains of TFRC KI mice.

Fig. 5.

Open in a new tab

(A) An ssAAV genome expressing human GBA1 was packaged into AAV9 or BI-hTFR1 and administered to TFRC KI transgenic mice at 1 × 1014 or 5 × 1012 vg/kg. AAV biodistribution and transduction were evaluated at three weeks post-injection. (B) AAV genome biodistribution to the brain and liver relative to AAV9 is shown (one-way ANOVA using AAV9 as the main comparison group with Sidak’s multiple comparison post-hoc test: **** and *** indicate p ≤ 0.0001 and ≤ 0.001, respectively; each data point represents an individual mouse, error bars indicate ± SEM). (C) Sagittal brain sections (top, scale bar = 1 mm) and representative images of the substantia nigra pars compacta (bottom, scale bar = 25 μm) from the mice in each group are shown. (D) GCase activity in brain and liver tissue homogenate, CSF, and serum are shown (one-way ANOVA using AAV9 as the main comparison group with Sidak’s multiple comparison post-hoc test: **** indicates p ≤ 0.0001; each data point represents an individual mouse, error bars indicate ± SEM).

To confirm the source of GBA expression, we quantified GBA1 mRNA and AAV genomes in the brains of TFRC KI mice treated with 1 × 1014 vg/kg of BI-hTFR1 carrying a codon-modified human GBA1. GBA1 was codon-modified to enable the selective detection of the human transgene but not endogenous mouse Gba1 mRNA. We observed greater GBA1 mRNA localization in the parenchyma than in the vasculature (fig. S9B and C) and greater AAV genome localization in parenchymal nuclei than in vascular nuclei at three weeks post-injection (fig. S9D and E). These data show that a substantial fraction of BI-hTFR1 was effectively transported across the vasculature.

To assess whether the intravenous administration of BI-hTFR1 might affect BBB integrity, we looked for barrier dysfunction with a biotin tracer leakage assay (36, 37). We assessed uninjected WT mice, the 1 × 1014 vg/kg BI-hTFR1:GBA1-treated TFRC KI cohort associated with figure S9B–E at three weeks post-administration, and a new 2 × 1013 vg/kg BI-hTFR1:GBA1-treated TFRC KI cohort at three hours, four days, or three weeks post-administration (fig. S10A). We chose this more therapeutically relevant dose based on the GBA1 overexpression observed in our initial study (Fig. 5) and observed once again that GBA1 RNA localization was largely extravascular (fig. S10B). The tracer was confined within vessels across the CNS (fig. S10C) with the exception of sparse spots that were observed at similar frequencies in all groups, including the uninjected mice (fig. S10D–F).

Throughout this study, we consistently produced and purified BI-hTFR1 with yields comparable to AAV9, and determined that BI-hTFR1 can be purified with commonly used affinity resins (fig. S11).

Discussion

We leveraged a well established mechanism—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 showing evidence of CNS entry (38) and efficacy in humans at doses predicted from TFRC KI mouse studies (19, 39, 40). We demonstrated that BI-hTFR1 binds TfR1 in the presence of holo-Tf, which 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 our confidence that BI-hTFR1 can perform well as a CNS-targeting human gene therapy vehicle. Furthermore, because our approach did not require the introduction or conjugation of bulky protein domains (41–44), we do not anticipate additional complexities in the manufacturing of BI-hTFR1 compared to AAV9, one of the top producing natural AAVs.

As compared to AAV9 and AAV2, BI-hTFR1 was more efficiently transported across a human brain endothelial cell layer and transduced 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 of engineered mouse BBB-crossing capsids that bind to mouse glycosylphosphatidylinositol (GPI)-anchored proteins (21, 45, 46). By engaging highly abundant CNS endothelial cell surface proteins as receptors, these capsids efficiently cross the BBB. Our study demonstrates that AAVs can be taken up and transported across the BBB by receptors other than GPI-anchored proteins.

A critical advantage of BI-hTFR1 is its known MOA, thus its species tropism is predictable and its function in patients can be predicted by evaluating human genetic variants of the receptor. This contrasts with capsids identified from conventional selections with unknown MOAs and unknown potential to translate to other species including humans. However, BI-hTFR1 exhibits human-specific TfR1 binding; thus, preclinical paths for investigational gene therapies using BI-hTFR1 will likely include on-target efficacy, biodistribution, and toxicology studies in TFRC KI mice, but not large animal models. Large animal studies would only inform off-target biodistribution, immunogenicity, and toxicity in the absence of the target receptor. Nonetheless, numerous human-specific biologics have reached the clinic and obtained FDA approval with (47–51) or without (52, 53) large animal off-target assessments.

A second advantage is that potential gene therapies leveraging BI-hTFR1 can be tested in mice possessing both human TFRC and disease-relevant mutation(s). In other words, the gene therapy product that would be delivered to patients can be tested preclinically without using a surrogate mouse BBB-crossing capsid. A limitation of our study is that we did not use BI-hTFR1 to ameliorate specific disease phenotypes. However, using a therapeutically relevant payload, we found that BI-hTFR1 could deliver 30 times the number of AAV:GBA1 genomes to the brains of TFRC KI mice and achieve greater increases in brain and CSF GCase activity compared to AAV9. Even at a 20-fold lower dose, GBA1 expression was detectable throughout the brains of BI-hTFR1:GBA1 treated mice.

Our results show that BI-hTFR1 can leverage human TfR1 to cross the BBB and efficiently deliver genes to neurons and glia throughout the CNS. These qualities strongly support the further exploration of BI-hTFR1 as a broadly applicable vehicle for CNS gene therapies.

Supplementary Material

Supplementary Material
Data S1

Materials and Methods

Figs. S1 to S11

Table S1

Data S1

References (54–60)

Acknowledgments:

We thank the members of the Deverman laboratory for discussions of the project, and particularly Saša Jereb for advice on performing RNAscope. We also thank Nadine Elowe and Tonia Aristotelous for helpful advice with the BLI studies; Richard Daneman, Chenghua Gu, Hannah Zucker, and Caterina Profaci for helpful discussions on blood brain-barrier leakage assays; and Tyler Caron and Nathan Chan for helpful discussions related to animal experiments.

Funding:

National Institutes of Health (NIH) Common Fund and the National Institute of Neurological Disorders and Stroke through the Somatic Cell Genome Engineering Consortium UG3NS111689 (BED)

Brain Initiative award funded through the National Institute of Mental Health UG3MH120096 (BED)

The Stanley Center for Psychiatric Research (BED)

Apertura Gene Therapy (BED)

Footnotes

Competing interests: B.E.D is a scientific founder and scientific advisory board member at Apertura Gene Therapy and a scientific advisory board member at Tevard Biosciences. B.E.D., A.J.B., K.Y.C., F.E.E., Q.H., J.W., and N.R.B.R. are named inventors on patent applications filed by the Broad Institute of MIT and Harvard related to this study. Remaining authors declare that they have no competing interests.

Data and materials availability:

All data are available in the manuscript or supplementary material. The code for generating counting RNA or DNA puncta and generating nuclei and vascular masks will be made available at https://github.com/vector-engineering/VasculatureMaskingAnalysis. BI-hTFR Rep-Cap and reporter construct sequences and plasmids will be made available on Addgene at the time of publication.

References and Notes

  • 1.Müller OJ, Kaul F, Weitzman MD, Pasqualini R, Arap W, Kleinschmidt JA, Trepel M, Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat. Biotechnol. 21, 1040–1046 (2003). [DOI] [PubMed] [Google Scholar]
  • 2.Deverman BE, Pravdo PL, Simpson BP, Kumar SR, Chan KY, Banerjee A, Wu W-L, Yang B, Huber N, Pasca SP, Gradinaru V, Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Nonnenmacher M, Wang W, Child MA, Ren X-Q, Huang C, Ren AZ, Tocci J, Chen Q, Bittner K, Tyson K, Pande N, Chung CH-Y, Paul SM, Hou J, Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning. Mol Ther Methods Clin Dev 20, 366–378 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hanlon KS, Meltzer JC, Buzhdygan T, Cheng MJ, Sena-Esteves M, Bennett RE, Sullivan TP, Razmpour R, Gong Y, Ng C, Nammour J, Maiz D, Dujardin S, Ramirez SH, Hudry E, Maguire CA, Selection of an Efficient AAV Vector for Robust CNS Transgene Expression. Mol Ther Methods Clin Dev 15, 320–332 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ajioka RS, Kaplan J, Intracellular pools of transferrin receptors result from constitutive internalization of unoccupied receptors. Proc. Natl. Acad. Sci. U. S. A. 83, 6445–6449 (1986). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Maxfield FR, McGraw TE, Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132 (2004). [DOI] [PubMed] [Google Scholar]
  • 7.Moos T, Morgan EH, Transferrin and transferrin receptor function in brain barrier systems. Cell. Mol. Neurobiol. 20, 77–95 (2000). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Pardridge WM, Eisenberg J, Yang J, Human blood-brain barrier transferrin receptor. Metabolism 36, 892–895 (1987). [DOI] [PubMed] [Google Scholar]
  • 9.Jefferies WA, Brandon MR, Hunt SV, Williams AF, Gatter KC, Mason DY, Transferrin receptor on endothelium of brain capillaries. Nature 312, 162–163 (1984). [DOI] [PubMed] [Google Scholar]
  • 10.Kida S, Koshimura Y, Yoden E, Yoshioka A, Morimoto H, Imakiire A, Tanaka N, Tanaka S, Mori A, Ito J, Inoue A, Yamamoto R, Minami K, Hirato T, Takahashi K, Sonoda H, Enzyme replacement with transferrin receptor-targeted α-L-iduronidase rescues brain pathology in mucopolysaccharidosis I mice. Mol Ther Methods Clin Dev 29, 439–449 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Hammond SM, Abendroth F, Goli L, Stoodley J, Burrell M, Thom G, Gurrell I, Ahlskog N, Gait MJ, Wood MJ, Webster CI, Antibody-oligonucleotide conjugate achieves CNS delivery in animal models for spinal muscular atrophy. JCI Insight 7 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kariolis MS, Wells RC, Getz JA, Kwan W, Mahon CS, Tong R, Kim DJ, Srivastava A, Bedard C, Henne KR, Giese T, Assimon VA, Chen X, Zhang Y, Solanoy H, Jenkins K, Sanchez PE, Kane L, Miyamoto T, Chew KS, Pizzo ME, Liang N, Calvert MEK, DeVos SL, Baskaran S, Hall S, Sweeney ZK, Thorne RG, Watts RJ, Dennis MS, Silverman AP, Zuchero YJY, Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys. Sci. Transl. Med. 12 (2020). [DOI] [PubMed] [Google Scholar]
  • 13.Rué L, Jaspers T, Degors IMS, Noppen S, Schols D, De Strooper B, Dewilde M, Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics. Pharmaceutics 15 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Yu YJ, Atwal JK, Zhang Y, Tong RK, Wildsmith KR, Tan C, Bien-Ly N, Hersom M, Maloney JA, Meilandt WJ, Bumbaca D, Gadkar K, Hoyte K, Luk W, Lu Y, Ernst JA, Scearce-Levie K, Couch JA, Dennis MS, Watts RJ, Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci. Transl. Med. 6, 261ra154 (2014). [DOI] [PubMed] [Google Scholar]
  • 15.van Lengerich B, Zhan L, Xia D, Chan D, Joy D, Park JI, Tatarakis D, Calvert M, Hummel S, Lianoglou S, Pizzo ME, Prorok R, Thomsen E, Bartos LM, Beumers P, Capell A, Davis SS, de Weerd L, Dugas JC, Duque J, Earr T, Gadkar K, Giese T, Gill A, Gnörich J, Ha C, Kannuswamy M, Kim DJ, Kunte ST, Kunze LH, Lac D, Lechtenberg K, Leung AW-S, Liang C-C, Lopez I, McQuade P, Modi A, Torres VO, Nguyen HN, Pesämaa I, Propson N, Reich M, Robles-Colmenares Y, Schlepckow K, Slemann L, Solanoy H, Suh JH, Thorne RG, Vieira C, Wind-Mark K, Xiong K, Zuchero YJY, Diaz D, Dennis MS, Huang F, Scearce-Levie K, Watts RJ, Haass C, Lewcock JW, Di Paolo G, Brendel M, Sanchez PE, Monroe KM, A TREM2-activating antibody with a blood-brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models. Nat. Neurosci. 26, 416–429 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Candelaria PV, Leoh LS, Penichet ML, Daniels-Wells TR, Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents. Front. Immunol. 12, 607692 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Barker SJ, Thayer MB, Kim C, Tatarakis D, Simon M, Dial RL, Nilewski L, Wells RC, Zhou Y, Afetian M, Chappell A, Chew KS, Chow J, Clemens A, Discenza CB, Dugas J, Dwyer C, Earr T, Ha C, Huynh D, Jayaraman S, Kwan W, Mahon C, Pizzo M, Roche E, Sanders L, Stergioulis A, Tong R, Tran H, Zuchero J, Estrada AA, Gadkar K, Koth CMM, Sanchez PE, Thorne RG, Watts RJ, Sandmann T, Kane L, Rigo F, Dennis MS, Lewcock JW, DeVos SL, Targeting Transferrin Receptor to Transport Antisense Oligonucleotides Across the Blood-Brain Barrier, bioRxiv (2023)p. 2023.04.25.538145. [DOI] [PubMed] [Google Scholar]
  • 18.Giugliani R, Martins AM, So S, Yamamoto T, Yamaoka M, Ikeda T, Tanizawa K, Sonoda H, Schmidt M, Sato Y, Iduronate-2-sulfatase fused with anti-hTfR antibody, pabinafusp alfa, for MPS-II: A phase 2 trial in Brazil. Mol. Ther. 29, 2378–2386 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Okuyama T, Eto Y, Sakai N, Nakamura K, Yamamoto T, Yamaoka M, Ikeda T, So S, Tanizawa K, Sonoda H, Sato Y, A Phase 2/3 Trial of Pabinafusp Alfa, IDS Fused with Anti-Human Transferrin Receptor Antibody, Targeting Neurodegeneration in MPS-II. Mol. Ther. 29, 671–679 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Smith L, Schapira AHV, GBA Variants and Parkinson Disease: Mechanisms and Treatments. Cells 11 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Huang Q, Chen AT, Chan KY, Sorensen H, Barry AJ, Azari B, Zheng Q, Beddow T, Zhao B, Tobey IG, Moncada-Reid C, Eid F-E, Walkey CJ, Ljungberg MC, Lagor WR, Heaney JD, Chan YA, Deverman BE, Targeting AAV vectors to the central nervous system by engineering capsid-receptor interactions that enable crossing of the blood-brain barrier. PLoS Biol. 21, e3002112 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. [See supplementary materials.]
  • 23.Ferrero S, Flores MD, Short C, Vazquez CA, Clark LE, Ziegenbein J, Zink S, Fuentes D, Payes C, Batto MV, Collazo M, García CC, Abraham J, Cordo SM, Rodriguez JA, Helguera G, Antibody-Based Inhibition of Pathogenic New World Hemorrhagic Fever Mammarenaviruses by Steric Occlusion of the Human Transferrin Receptor 1 Apical Domain. J. Virol. 95, e0186820 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Williams J, Moreton K, The distribution of iron between the metal-binding sites of transferrin human serum. Biochem. J 185, 483–488 (1980). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Balzer V, Poc P, Puris E, Martin S, Aliasgari M, Auriola S, Fricker G, Re-evaluation of the hCMEC/D3 based in vitro BBB model for ABC transporter studies. Eur. J. Pharm. Biopharm. 173, 12–21 (2022). [DOI] [PubMed] [Google Scholar]
  • 26.Di Pasquale G, Chiorini JA, AAV transcytosis through barrier epithelia and endothelium. Mol. Ther. 13, 506–516 (2006). [DOI] [PubMed] [Google Scholar]
  • 27.Merkel SF, Andrews AM, Lutton EM, Mu D, Hudry E, Hyman BT, Maguire CA, Ramirez SH, Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells. J. Neurochem. 140, 216–230 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Song R, Pekrun K, Khan TA, Zhang F, Paşca SP, Kay MA, Selection of rAAV vectors that cross the human blood-brain barrier and target the central nervous system using a transwell model. Mol Ther Methods Clin Dev 27, 73–88 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Nonnenmacher ME, Cintrat J-C, Gillet D, Weber T, Syntaxin 5-dependent retrograde transport to the trans-Golgi network is required for adeno-associated virus transduction. J. Virol. 89, 1673–1687 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Navaroli DM, Bellvé KD, Standley C, Lifshitz LM, Cardia J, Lambright D, Leonard D, Fogarty KE, Corvera S, Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis. Proc. Natl. Acad. Sci. U. S. A. 109, E471–80 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Haqqani AS, Delaney CE, Brunette E, Baumann E, Farrington GK, Sisk W, Eldredge J, Ding W, Tremblay T-L, Stanimirovic DB, Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier. J. Cereb. Blood Flow Metab. 38, 727–740 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, Lowes L, Alfano L, Berry K, Church K, Kissel JT, Nagendran S, L’Italien J, Sproule DM, Wells C, Cardenas JA, Heitzer MD, Kaspar A, Corcoran S, Braun L, Likhite S, Miranda C, Meyer K, Foust KD, Burghes AHM, Kaspar BK, Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N. Engl. J. Med. 377, 1713–1722 (2017). [DOI] [PubMed] [Google Scholar]
  • 33.Oftedal L, Maple-Grødem J, Dalen I, Tysnes O-B, Pedersen KF, Alves G, Lange J, Association of CSF Glucocerebrosidase Activity With the Risk of Incident Dementia in Patients With Parkinson Disease. Neurology 100, e388–e395 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Rocha EM, Smith GA, Park E, Cao H, Brown E, Hayes MA, Beagan J, McLean JR, Izen SC, Perez-Torres E, Hallett PJ, Isacson O, Glucocerebrosidase gene therapy prevents α-synucleinopathy of midbrain dopamine neurons. Neurobiol. Dis. 82, 495–503 (2015). [DOI] [PubMed] [Google Scholar]
  • 35.Seidel K, Mahlke J, Siswanto S, Krüger R, Heinsen H, Auburger G, Bouzrou M, Grinberg LT, Wicht H, Korf H-W, den Dunnen W, Rüb U, The brainstem pathologies of Parkinson’s disease and dementia with Lewy bodies. Brain Pathol. 25, 121–135 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Daneman R, Zhou L, Kebede AA, Barres BA, Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562–566 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Ayloo S, Lazo CG, Sun S, Zhang W, Cui B, Gu C, Pericyte-to-endothelial cell signaling via vitronectin-integrin regulates blood-CNS barrier. Neuron 110, 1641–1655.e6 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Grimm HP, Schumacher V, Schäfer M, Imhof-Jung S, Freskgård P-O, Brady K, Hofmann C, Rüger P, Schlothauer T, Göpfert U, Hartl M, Rottach S, Zwick A, Seger S, Neff R, Niewoehner J, Janssen N, Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans. MAbs 15, 2261509 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Okuyama T, Eto Y, Sakai N, Minami K, Yamamoto T, Sonoda H, Yamaoka M, Tachibana K, Hirato T, Sato Y, Iduronate-2-Sulfatase with Anti-human Transferrin Receptor Antibody for Neuropathic Mucopolysaccharidosis II: A Phase 1/2 Trial. Mol. Ther. 27, 456–464 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Sonoda H, Morimoto H, Yoden E, Koshimura Y, Kinoshita M, Golovina G, Takagi H, Yamamoto R, Minami K, Mizoguchi A, Tachibana K, Hirato T, Takahashi K, A Blood-Brain-Barrier-Penetrating Anti-human Transferrin Receptor Antibody Fusion Protein for Neuronopathic Mucopolysaccharidosis II. Mol. Ther. 26, 1366–1374 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ponnazhagan S, Mahendra G, Kumar S, Thompson JA, Castillas M Jr, Conjugate-based targeting of recombinant adeno-associated virus type 2 vectors by using avidin-linked ligands. J. Virol. 76, 12900–12907 (2002). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Münch RC, Janicki H, Völker I, Rasbach A, Hallek M, Büning H, Buchholz CJ, Displaying high-affinity ligands on adeno-associated viral vectors enables tumor cell-specific and safe gene transfer. Mol. Ther. 21, 109–118 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Eichhoff AM, Börner K, Albrecht B, Schäfer W, Baum N, Haag F, Körbelin J, Trepel M, Braren I, Grimm D, Adriouch S, Koch-Nolte F, Nanobody-Enhanced Targeting of AAV Gene Therapy Vectors. Mol Ther Methods Clin Dev 15, 211–220 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hamann MV, Beschorner N, Vu X-K, Hauber I, Lange UC, Traenkle B, Kaiser PD, Foth D, Schneider C, Büning H, Rothbauer U, Hauber J, Improved targeting of human CD4+ T cells by nanobody-modified AAV2 gene therapy vectors. PLoS One 16, e0261269 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Huang Q, Chan KY, Tobey IG, Chan YA, Poterba T, Boutros CL, Balazs AB, Daneman R, Bloom JM, Seed C, Deverman BE, Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids. PLoS One 14, e0225206 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Shay TF, Sullivan EE, Ding X, Chen X, Ravindra Kumar S, Goertsen D, Brown D, Crosby A, Vielmetter J, Borsos M, Wolfe DA, Lam AW, Gradinaru V, Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors. Sci Adv 9, eadg6618 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.BLA 761035/ELOTUZUMAB/EMPLICITI, FDA. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/761035Orig1s000CrossR.pdf.
  • 48.BLA761244/SPESOLIMAB-SBZO/SPEVIGO, FDA. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2022/761244Orig1s000MultidisciplineR.pdf.
  • 49.BLA 761342/TALQUETAMAB-TGVS/TALVEY, FDA. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2023/761342Orig1s000MultidisciplineR.pdf.
  • 50.BLA 761113/ISATUXIMAB-IRFC/SARCLISA, FDA. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2020/761113Orig1s000MultidisciplineR.pdf.
  • 51.BLA 761108/RAVULIZUMAB-CWVZ/ULTOMIRIS, FDA. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2018/761108Orig1s000MultidisciplineR.pdf.
  • 52.BLA # 761142 /INEBILIZUMAB-CDON/UPLIZNA, FDA. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2020/761142Orig1s000PharmR.pdf.
  • 53.BLA 761228/TEBENTAFUSP-TEBN/KIMMTRAK, FDA. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2022/761228Orig1s000MultidisciplineR.pdf.
  • 54.Errington WJ, Bruncsics B, Sarkar CA, Mechanisms of noncanonical binding dynamics in multivalent protein-protein interactions. Proc. Natl. Acad. Sci. U. S. A. 116, 25659–25667 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Schoeffler AJ, Helgason E, Popovych N, Dueber EC, Diagnosing and mitigating method-based avidity artifacts that confound polyubiquitin-binding assays. Biophys Rep (N Y) 1, 100033 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Eid F-E, Chen AT, Chan KY, Huang Q, Zheng Q, Tobey IG, Pacouret S, Brauer PP, Keyes C, Powell M, Johnston J, Zhao B, Lage K, Tarantal AF, Chan YA, Deverman BE, Systematic multi-trait AAV capsid engineering for efficient gene delivery, bioRxiv (2022)p. 2022.12.22.521680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Challis RC, Ravindra Kumar S, Chan KY, Challis C, Beadle K, Jang MJ, Kim HM, Rajendran PS, Tompkins JD, Shivkumar K, Deverman BE, Gradinaru V, Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat. Protoc. 14, 379–414 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Lim NK-H, Moestrup V, Zhang X, Wang W-A, Møller A, Huang F-D, An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice. J. Vis. Exp, doi: 10.3791/56774 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Berg S, Kutra D, Kroeger T, Straehle CN, Kausler BX, Haubold C, Schiegg M, Ales J, Beier T, Rudy M, Eren K, Cervantes JI, Xu B, Beuttenmueller F, Wolny A, Zhang C, Koethe U, Hamprecht FA, Kreshuk A, ilastik: interactive machine learning for (bio)image analysis. Nat. Methods 16, 1226–1232 (2019). [DOI] [PubMed] [Google Scholar]
  • 60.Stirling DR, Swain-Bowden MJ, Lucas AM, Carpenter AE, Cimini BA, Goodman A, CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics 22, 433 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material
NIHMS2121597-supplement-Supplementary_Material.pdf (17.9MB, pdf)
Data S1
NIHMS2121597-supplement-Data_S1.xlsx (6.1MB, xlsx)

Data Availability Statement

All data are available in the manuscript or supplementary material. The code for generating counting RNA or DNA puncta and generating nuclei and vascular masks will be made available at https://github.com/vector-engineering/VasculatureMaskingAnalysis. BI-hTFR Rep-Cap and reporter construct sequences and plasmids will be made available on Addgene at the time of publication.

RESOURCES