Danica B. Stanimirovic

Development of therapies and pharmacological management of central nervous system diseases is significantly hampered by the presence of the highly selective blood–brain barrier (BBB). Receptor-mediated transcytosis (RMT) has proven to be a robust mechanism for delivering a wide variety of therapeutic modalities across the BBB. The present study describes the use of a neurotensin-based pharmacodynamic model to evaluate the brain penetrating capacity of FC5, a well-characterized RMT shuttle. Here we produce fusion molecules of FC5 with human Fc and neurotensin and show that intact but not C-terminally clipped neurotensin proteins containing either FC5 (FC5Fc-neurotensin) or a non-BBB-crossing control (A20.1Fc-neurotensin) retain the ability to activate neurotensin receptor type 1. In in vivo studies, intravenous administration of FC5Fc-neurotensin, but not neurotensin alone or A20.1Fc-neurotensin, induced a drop in the core body temperature in both rats and mice. We further extended these findings to non-human primates, demonstrating the ability of FC5 to cross the BBB in this species. Collectively, our results further support the potential of FC5 in delivering payloads across the BBB. The neurotensin model of hypothermia is also validated as a pharmacodynamic approach for assessing brain delivery, albeit with some necessary considerations into the molecular characterization of fusion constructs before in vivo evaluation.

Genetic studies have identified the voltage‐gated sodium channel 1.7 (Na_v1.7) as pain target. Due to the ineffectiveness of small molecules and monoclonal antibodies as therapeutics for pain, single‐domain antibodies (V_HHs) are developed against the human Na_v1.7 (hNa_v1.7) using a novel antigen presentation strategy. A 70 amino‐acid peptide from the hNa_v1.7 protein is identified as a target antigen. A recombinant version of this peptide is grafted into the complementarity determining region 3 (CDR3) loop of an inert V_HH in order to maintain the native 3D conformation of the peptide. This antigen is used to isolate one V_HH able to i) bind hNa_v1.7, ii) slow the deactivation of hNa_v1.7, iii) reduce the ability of eliciting action potentials in nociceptors, and iv) reverse hyperalgesia in in vivo rat and mouse models. This V_HH exhibits the potential to be developed as a therapeutic capable of suppressing pain. This novel antigen presentation strategy can be applied to develop biologics against other difficult targets such as ion channels, transporters and GPCRs.

Background: Vascular cognitive impairment and dementia (VCID), the second most prevalent of the age‐related dementias, develops as a consequence of various types of cerebrovascular insults that damage brain function (Corriveau et al. 2016). Accumulating lines of evidence point to a link between VCID, both sporadic (Kim et al. 2006) and genetic (Hara et al. 2009, Zellner et al. 2018) forms, and the transforming growth factor beta (TGFB) family signaling. Transgenic mice overexpressing a constitutively active form of TGFβ1 in the brain (TGF mice) recap the cerebrovascular pathology seen in VCID (Wyss‐Coray et al. 2000, Tong et al. 2015), and develop VCID when submitted to a comorbid cardiovascular risk factor for dementia (Trigiani et al. 2020). Our aim was to characterize the cerebrovascular proteome of TGF mice using mass spectrometry‐based quantitative proteomics.Method: Eighteen, 6‐month‐old‐TGF and ‐wildtype (WT) mice (N = 9/group) were transcardially perfused and pial arteries harvested under a dissecting microscope. Arterial proteins were extracted, trypsin‐digested, fractionated by strong cation exchange (SCX, gel‐free method) and analyzed by nanoLC‐MS/MS using nanoAcquity UPLC and ESI‐LTQ Orbitrap. For total and/or differentially‐expressed proteins (≥2‐fold change, p≤0.01) we i) performed characterization of proteins, and demonstrated presence of ii) protein RNA‐transcript in mouse and human brain vascular cells using transcriptomics datasets, and iii) identified proteins present in human‐extracellular‐vesicles (EVs) using Vesiclepedia.Result: We identified 3602 proteins in brain vessels of WT and TGF mice, including canonical vascular proteins (e.g. Claudin‐5), and 103 direct (N = 103) and indirect (N = 1,942) interactors of TGFβ1 (Fig1). We also identified 83 proteins demonstrating significantly altered levels in TGF mice. Level dysregulation in these proteins point to perturbations in brain vessel vasomotricity, remodeling, and inflammation. We further demonstrated that several of the differentially‐expressed mouse proteins are i) expressed in the human brain vasculature, and ii) found as cargo proteins in EVs.Conclusion: We characterized the deleterious impact of TGFβ1 overproduction on the cerebrovascular proteome. Given the growing popularity of extracellular vesicles in blood as a novel and minimally invasive biomarker discovery platform for the age‐related dementias, including VCID, several of the proteins identified by us can serve as protein biomarkers in human.

Cerebrovascular pathology that involves altered protein levels (or signaling) of the transforming growth factor beta (TGFβ) family has been associated with various forms of age-related dementias, including Alzheimer disease (AD) and vascular cognitive impairment and dementia (VCID). Transgenic mice overexpressing TGFβ1 in the brain (TGF mice) recapitulate VCID-associated cerebrovascular pathology and develop cognitive deficits in old age or when submitted to comorbid cardiovascular risk factors for dementia. We characterized the cerebrovascular proteome of TGF mice using mass spectrometry (MS)-based quantitative proteomics. Cerebral arteries were surgically removed from 6-month-old-TGF and wild-type mice, and proteins were extracted and analyzed by gel-free nanoLC-MS/MS. We identified 3602 proteins in brain vessels, with 20 demonstrating significantly altered levels in TGF mice. For total and/or differentially expressed proteins (p ≤ 0.01, ≥ 2-fold change), using multiple databases, we (a) performed protein characterization, (b) demonstrated the presence of their RNA transcripts in both mouse and human cerebrovascular cells, and (c) demonstrated that several of these proteins were present in human extracellular vesicles (EVs) circulating in blood. Finally, using human plasma, we demonstrated the presence of several of these proteins in plasma and plasma EVs. Dysregulated proteins point to perturbed brain vessel vasomotricity, remodeling, and inflammation. Given that blood-isolated EVs are novel, attractive, and a minimally invasive biomarker discovery platform for age-related dementias, several proteins identified in this study can potentially serve as VCID markers in humans.

A cerebrovascular pathology that involves altered protein levels or signaling of the transforming growth factor beta (TGFβ) family has been associated with various forms of dementia, including Alzheimer disease (AD) and vascular cognitive impairment and dementia (VCID). Transgenic mice overexpressing TGFβ1 in the brain (TGF mice) recap VCID-associated cerebrovascular pathology and develop cognitive deficits in old age or when submitted to comorbid cardiovascular risk-factors for dementia. Here, we characterized the cerebrovascular proteome of TGF mice using mass-spectrometry (MS) based quantitative proteomics. Cerebral arteries were surgically removed from 6-month-old-TGF and wild-type mice, proteins extracted and analyzed by gel-free nanoLC-MS/MS. We identified 3,602 proteins in brain vessels, with 20 demonstrating robust altered levels in TGF mice. For total and/or differentially-expressed proteins ( p ≤0.01, ≥2-fold change), using multiple databases, we performed protein characterization, and identified proteins demonstrating RNA-transcripts in both mouse and human cerebrovascular cells, and known to be present in human-extracellular-vesicles (EVs). Dysregulated proteins point to perturbed brain vessel vasomotricity, remodeling, and inflammation. Given that blood-isolated EVs are novel, attractive and a minimally invasive biomarker discovery platform for the age-related dementias, several proteins identified in this study can potentially serve as VCID markers in humans.

Lipoprotein lipase deficiency (LPLD) results from mutations within the lipoprotein lipase (LPL) gene that lead to a complete lack of catalytically active LPL protein. Glybera was one of the first adeno-associated virus (AAV) gene replacement therapy to receive European Medicines Agency regulatory approval for the treatment of LPLD. However, Glybera is no longer marketed potentially due to a combination of economical, manufacturing and vector related issues. The aim of this study was to develop a more efficacious AAV gene therapy vector for LPLD. Following preclinical biodistribution, efficacy and non-GLP toxicity studies with novel AAV1 and AAV8-based vectors in mice, we identified AAV8 pVR59. AAV8 pVR59 delivered a codon optimized, human gain-of-function hLPLS447X transgene driven by a CAG promoter in an AAV8 capsid. AAV8 pVR59 was significantly more efficacious, at 10- to 100-fold lower doses, compared to an AAV1 vector based on Glybera, when delivered intramuscularly or intravenously, respectively, in mice with LPLD. Efficient gene transfer was observed within the injected skeletal muscle and liver following delivery of AAV8 pVR59, with long-term correction of LPLD phenotypes, including normalization of plasma triglycerides and lipid tolerance, for up to 6 months post-treatment. While intramuscular delivery of AAV8 pVR59 was well tolerated, intravenous administration augmented liver pathology. These results highlight the feasibility of developing a superior AAV vector for the treatment of LPLD and provide critical insight for initiating studies in larger animal models. The identification of an AAV gene therapy vector that is more efficacious at lower doses, when paired with recent advances in production and manufacturing technologies, will ultimately translate to increased safety and accessibility for patients.

Background: ATP-binding cassette (ABC) transporters comprise a superfamily of genes encoding membrane proteins with nucleotide-binding domains (NBD). These transporters, including drug efflux across the blood–brain barrier (BBB), carry a variety of substrates through plasma membranes against substrate gradients, fueled by hydrolyzing ATP. The expression patterns/enrichment of ABC transporter genes in brain microvessels compared to peripheral vessels and tissues are largely uncharacterized. Methods: In this study, the expression patterns of ABC transporter genes in brain microvessels, peripheral tissues (lung, liver and spleen) and lung vessels were investigated using RNA-seq and Wes^TM analyses in three species: human, mouse and rat. Results: The study demonstrated that ABC drug efflux transporter genes (including ABCB1, ABCG2, ABCC4 and ABCC5) were highly expressed in isolated brain microvessels in all three species studied; the expression of ABCB1, ABCG2, ABCC1, ABCC4 and ABCC5 was generally higher in rodent brain microvessels compared to those of humans. In contrast, ABCC2 and ABCC3 expression was low in brain microvessels, but high in rodent liver and lung vessels. Overall, most ABC transporters (with the exception of drug efflux transporters) were enriched in peripheral tissues compared to brain microvessels in humans, while in rodent species, additional ABC transporters were found to be enriched in brain microvessels. Conclusions: This study furthers the understanding of species similarities and differences in the expression patterns of ABC transporter genes; this is important for translational studies in drug development. In particular, CNS drug delivery and toxicity may vary among species depending on their unique profiles of ABC transporter expression in brain microvessels and BBB.

The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood–brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.

The blood‐brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non‐invasive technologies targeting receptor‐mediated pathways have been developed. In this study, three single‐domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin‐like growth factor‐1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV‐ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb‐mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation. Compared to the brains perfused with the negative control A20.1‐mFc, the brains perfused with anti‐IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2‐fold, p < .01) and the distribution volume (~4‐fold, p < .01). The concentration curve for IGF1R4‐mFc demonstrated a linear accumulation plateauing at approximately 400 µg (~1 µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post‐ISBP confirmed the IGF1R4‐mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1‐mFc after a 5‐min perfusion protocol. Systemic administration of IGF1R4‐mFc fused with the non‐BBB crossing analgesic peptide galanin (2 and 5 mg/kg) induced a dose‐dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti‐IGF1R sdAbs showed receptor‐mediated brain uptake with pharmacologically effective parenchymal delivery of non‐permeable neuroactive peptides.