Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus.

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models.

Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The stimulator of interferon genes (STING) DNA sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties. Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils, and natural killer (NK) populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, chimeric antigen receptor T cells, NK therapies, and immune checkpoint blockade.

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers.

BACKGROUND: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). METHODS: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. RESULTS: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. CONCLUSION: GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.

FASN Is a Biomarker Enriched in Malignant Glioma-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are known for their important role in cancer progression and hold considerable potential as a source for tumor biomarkers. However, purification of tumor-specific EVs from patient plasma is still an urgent unmet need due to contamination by normal host cell-derived EVs, that results in compromised analytical sensitivity. Here we identified fatty acid synthase (FASN), a key lipogenic enzyme which is highly expressed in malignant glioma cells, to be elevated in CD63- and CD81-positive EVs in glioma patient plasma samples, opening vital opportunities to sort brain tumor-specific EVs.

Characterization of glioblastoma in an orthotopic mouse model with magnetic resonance elastography.

Glioblastoma (GBM) is the most common primary brain tumor. It is highly malignant and has a correspondingly poor prognosis. Diagnosis and monitoring are mainly accomplished with MRI, but remain challenging in some cases. Therefore, complementary methods for tumor detection and characterization would be beneficial. Using magnetic resonance elastography (MRE), we performed a longitudinal study of the biomechanical properties of intracranially implanted GBM in mice and compared the results to histopathology. The biomechanical parameters of viscoelastic modulus, shear wave speed and phase angle were significantly lower in tumors compared with healthy brain tissue and decreased over time with tumor progression. Moreover, some MRE parameters revealed sub-regions at later tumor stages, which were not easily detectable on anatomical MRI images. Comparison with histopathology showed that softer tumor regions contained necrosis and patches of viable tumor cells. In contrast, areas of densely packed tumor cells and blood vessels identified with histology coincided with higher values of viscoelastic modulus and shear wave speed. Interestingly, the phase angle was independent from these anatomical variations. In summary, MRE depicted longitudinal and morphological changes in GBM and may prove valuable for tumor characterization in patients.

MicroRNA-451 regulates LKB1/AMPK signaling and allows adaptation to metabolic stress in glioma cells.

To sustain tumor growth, cancer cells must be able to adapt to fluctuations in energy availability. We have identified a single microRNA that controls glioma cell proliferation, migration, and responsiveness to glucose deprivation. Abundant glucose allows relatively high miR-451 expression, promoting cell growth. In low glucose, miR-451 levels decrease, slowing proliferation but enhancing migration and survival. This allows cells to survive metabolic stress and seek out favorable growth conditions. In glioblastoma patients, elevated miR-451 is associated with shorter survival. The effects of miR-451 are mediated by LKB1, which it represses through targeting its binding partner, CAB39 (MO25 alpha). Overexpression of miR-451 sensitized cells to glucose deprivation, suggesting that its downregulation is necessary for robust activation of LKB1 in response to metabolic stress. Thus, miR-451 is a regulator of the LKB1/AMPK pathway, and this may represent a fundamental mechanism that contributes to cellular adaptation in response to altered energy availability.

Perfluoroarene-Based Peptide Macrocycles to Enhance Penetration Across the Blood-Brain Barrier.

Here we describe the utility of peptide macrocyclization through perfluoroaryl-cysteine SNAr chemistry to improve the ability of peptides to cross the blood-brain barrier. Multiple macrocyclic analogues of the peptide transportan-10 were investigated that displayed increased uptake in two different cell lines and improved proteolytic stability. One of these analogues (M13) exhibited substantially increased delivery across a cellular spheroid model of the blood-brain barrier. Through ex vivo imaging of mouse brains, we demonstrated that this perfluoroarene-based macrocycle of TP10 exhibits increased penetration of the brain parenchyma following intravenous administration in mice. Finally, we evaluated macrocyclic analogues of the BH3 domain of the BIM protein to assess if our approach would be applicable to a peptide of therapeutic interest. We identified a BIM BH3 analogue that showed increased penetration of the brain tissue in mice.

Cytomegalovirus and glioblastoma; controversies and opportunities.

One of the more polarized ongoing debates in the brain tumor field over recent years has centered on the association of cytomegalovirus (CMV) with glioblastoma. Several laboratories have reported the presence of CMV antigens in glioblastoma patient specimens, whereas others have failed to detect them. CMV genomic DNA and mRNAs have been detected by PCR, but not in next-generation sequencing studies. CMV promotes high grade glioma progression in a mouse genetic model, and many CMV proteins promote cancer hallmarks in vitro, but actively replicating virus has not been isolated from tumor samples. A consensus is gradually emerging in which the presence of CMV antigens in glioblastoma is increasingly accepted. However, it remains challenging to understand this mechanistically due to the low levels of CMV nucleic acids and the absence of viral replication observed in tumors thus far. Nonetheless, these observations have inspired the development of novel therapeutic approaches based on anti-viral drugs and immunotherapy. The potential benefit of valganciclovir in glioblastoma has generated great interest, but efficacy remains to be established in a randomized trial. Also, early stage immunotherapy trials targeting CMV have shown promise. In the near future we will know more answers to these questions, and although areas of controversy may remain, and the mechanisms and roles of CMV in tumor growth are yet to be clearly defined, this widespread virus may have created important new therapeutic concepts and opportunities for the treatment of glioblastoma.

N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) promote growth and inhibit differentiation of glioma stem-like cells.

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.

Triacetin-based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma.

Cancer is associated with epigenetic (i.e., histone hypoacetylation) and metabolic (i.e., aerobic glycolysis) alterations. Levels of N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate, are reduced in glioma; yet, few studies have investigated acetate as a potential therapeutic agent. This preclinical study sought to test the efficacy of the food additive Triacetin (glyceryl triacetate, GTA) as a novel therapy to increase acetate bioavailability in glioma cells. The growth-inhibitory effects of GTA, compared to the histone deacetylase inhibitor Vorinostat (SAHA), were assessed in established human glioma cell lines (HOG and Hs683 oligodendroglioma, U87 and U251 glioblastoma) and primary tumor-derived glioma stem-like cells (GSCs), relative to an oligodendrocyte progenitor line (Oli-Neu), normal astrocytes, and neural stem cells (NSCs) in vitro. GTA was also tested as a chemotherapeutic adjuvant with temozolomide (TMZ) in orthotopically grafted GSCs. GTA-induced cytostatic growth arrest in vitro comparable to Vorinostat, but, unlike Vorinostat, GTA did not alter astrocyte growth and promoted NSC expansion. GTA alone increased survival of mice engrafted with glioblastoma GSCs and potentiated TMZ to extend survival longer than TMZ alone. GTA was most effective on GSCs with a mesenchymal cell phenotype. Given that GTA has been chronically administered safely to infants with Canavan disease, a leukodystrophy due to ASPA mutation, GTA-mediated acetate supplementation may provide a novel, safe chemotherapeutic adjuvant to reduce the growth of glioma tumors, most notably the more rapidly proliferating, glycolytic and hypoacetylated mesenchymal glioma tumors.

Intracranial CAR-T cell delivery in glioblastoma patients.

Chimeric antigen receptor (CAR)-T cell therapy is emerging as a promising approach for improving outcomes in high-grade glioma. Here, we highlight three recent studies that reported safety and feasibility of intracranial CAR-T cell administration in patients with glioblastoma (GBM) as well as preliminary evidence of potential responses, supporting further investigations of this approach.

Clinical implications of cytomegalovirus in glioblastoma progression and therapy.

Glioblastoma (GBM) is one of the deadliest brain cancers with a median survival of only 15 months. This poor prognosis has prompted exploration of novel therapeutic targets for GBM patients. Human cytomegalovirus (HCMV) has been implicated in GBM; however, its impact remains poorly defined, and there is conflicting data over the presence of HCMV in tumors. Nonetheless, clinical trials targeting HCMV have shown promising initial data, and evidence suggests that HCMV may negatively impact GBM patient survival by multiple mechanisms including changes in GBM cell behavior and the tumor microenvironment (TME) that potentiate tumor progression as well as therapy-induced virus reactivation. Moreover, HCMV has many effects on host immunity that could impact tumor behavior by altering the TME, which are largely unexplored. The goal of this review is to describe these potential interactions between HCMV and GBM. Better understanding of these processes may allow the development of new therapeutic modalities to improve GBM patient outcomes.

Modulation of blood-tumor barrier transcriptional programs improves intra-tumoral drug delivery and potentiates chemotherapy in GBM.

Glioblastoma (GBM) is the most common malignant primary brain tumor. GBM has an extremely poor prognosis and new treatments are badly needed. Efficient drug delivery to GBM is a major obstacle as the blood-brain barrier (BBB) prevents passage of the majority of cancer drugs into the brain. It is also recognized that the blood-brain tumor barrier (BTB) in the growing tumor represents a challenge. The BTB is heterogeneous and poorly characterized, but similar to the BBB it can prevent therapeutics from reaching effective intra-tumoral doses, dramatically hindering their potential. Here, we identified a 12-gene signature associated with the BTB, with functions related to vasculature development, morphogenesis and cell migration. We identified CDH5 as a core molecule in this set and confirmed its over-expression in GBM vasculature using spatial transcriptomics of GBM patient specimens. We found that the indirubin-derivative, 6-bromoindirubin acetoxime (BIA), could downregulate CDH5 and other BTB signature genes, causing endothelial barrier disruption in endothelial monolayers and BBB 3D spheroids in vitro. Treatment of tumor-bearing mice with BIA enabled increased intra-tumoral accumulation of the BBB non-penetrant chemotherapeutic drug cisplatin and potentiated cisplatin-mediated DNA damage by targeting DNA repair pathways. Finally, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved the efficacy of cisplatin in patient-derived GBM xenograms and prolonged their survival. Overall, our work reveals potential targets at the BTB for improved chemotherapy delivery and the bifunctional properties of BIA as a BTB modulator and potentiator of chemotherapy, supporting its further development.

Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models.

Rationale: CAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell's genome, thereby inflicting immunogenic cancer cell death. While CAN-2409's immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations. Methods: We performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors. Results: PCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12. Conclusion: CAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

PPRX-1701, a nanoparticle formulation of 6'-bromoindirubin acetoxime, improves delivery and shows efficacy in preclinical GBM models.

Derivatives of the Chinese traditional medicine indirubin have shown potential for the treatment of cancer through a range of mechanisms. This study investigates the impact of 6'-bromoindirubin-3'-acetoxime (BiA) on immunosuppressive mechanisms in glioblastoma (GBM) and evaluates the efficacy of a BiA nanoparticle formulation, PPRX-1701, in immunocompetent mouse GBM models. Transcriptomic studies reveal that BiA downregulates immune-related genes, including indoleamine 2,3-dioxygenase 1 (IDO1), a critical enzyme in the tryptophan-kynurenine-aryl hydrocarbon receptor (Trp-Kyn-AhR) immunosuppressive pathway in tumor cells. BiA blocks interferon-γ (IFNγ)-induced IDO1 protein expression in vitro and enhances T cell-mediated tumor cell killing in GBM stem-like cell co-culture models. PPRX-1701 reaches intracranial murine GBM and significantly improves survival in immunocompetent GBM models in vivo. Our results indicate that BiA improves survival in murine GBM models via effects on important immunotherapeutic targets in GBM and that it can be delivered efficiently via PPRX-1701, a nanoparticle injectable formulation of BiA.

Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature.

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity.

Pro-inflammatory T cells mediate autoimmune demyelination in multiple sclerosis. However, the factors driving their development and multiple sclerosis susceptibility are incompletely understood. We investigated how micro-RNAs, newly described as post-transcriptional regulators of gene expression, contribute to pathogenic T-cell differentiation in multiple sclerosis. miR-128 and miR-27b were increased in naïve and miR-340 in memory CD4(+) T cells from patients with multiple sclerosis, inhibiting Th2 cell development and favouring pro-inflammatory Th1 responses. These effects were mediated by direct suppression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and interleukin-4 (IL4) expression, resulting in decreased GATA3 levels, and a Th2 to Th1 cytokine shift. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic potential of myelin-specific T cells in experimental autoimmune encephalomyelitis. In addition, treatment of multiple sclerosis patient T cells with oligonucleotide micro-RNA inhibitors led to the restoration of Th2 responses. These data illustrate the biological significance and therapeutic potential of these micro-RNAs in regulating T-cell phenotypes in multiple sclerosis.

Metabolic Reprogramming of Glioblastoma Cells during HCMV Infection Induces Secretome-Mediated Paracrine Effects in the Microenvironment.

Glioblastoma (GBM) is an aggressive primary central nervous system neoplasia with limited therapeutic options and poor prognosis. Following reports of cytomegalovirus (HCMV) in GBM tumors, the anti-viral drug Valganciclovir was administered and found to significantly increase the longevity of GBM patients. While these findings suggest a role for HCMV in GBM, the relationship between them is not clear and remains controversial. Treatment with anti-viral drugs may prove clinically useful; however, their results do not explain the underlying mechanism between HCMV infection and GBM progression. We hypothesized that HCMV infection would metabolically reprogram GBM cells and that these changes would allow for increased tumor progression. We infected LN-18 GBM cells and employed a Seahorse Bioanalyzer to characterize cellular metabolism. Increased mitochondrial respiration and glycolytic rates were observed following infection. These changes were accompanied by elevated production of reactive oxygen species and lactate. Due to lactate's numerous tumor-promoting effects, we examined the impact of paracrine signaling of HCMV-infected GBM cells on uninfected stromal cells. Our results indicated that, independent of viral transmission, the secretome of HCMV-infected GBM cells was able to alter the expression of key metabolic proteins and epigenetic markers. This suggests a mechanism of action where reprogramming of GBM cells alters the surrounding tumor microenvironment to be permissive to tumor progression in a manner akin to the Reverse-Warburg Effect. Overall, this suggests a potential oncomodulatory role for HCMV in the context of GBM.

Inflammasome activation: from molecular mechanisms to autoinflammation.

Inflammasomes are assembled by innate immune sensors that cells employ to detect a range of danger signals and respond with pro-inflammatory signalling. Inflammasomes activate inflammatory caspases, which trigger a cascade of molecular events with the potential to compromise cellular integrity and release the IL-1ß and IL-18 pro-inflammatory cytokines. Several molecular mechanisms, working in concert, ensure that inflammasome activation is tightly regulated; these include NLRP3 post-translational modifications, ubiquitination and phosphorylation, as well as single-domain proteins that competitively bind to key inflammasome components, such as the CARD-only proteins (COPs) and PYD-only proteins (POPs). These diverse regulatory systems ensure that a suitable level of inflammation is initiated to counteract any cellular insult, while simultaneously preserving tissue architecture. When inflammasomes are aberrantly activated can drive excessive production of pro-inflammatory cytokines and cell death, leading to tissue damage. In several autoinflammatory conditions, inflammasomes are aberrantly activated with subsequent development of clinical features that reflect the degree of underlying tissue and organ damage. Several of the resulting disease complications may be successfully controlled by anti-inflammatory drugs and/or specific cytokine inhibitors, in addition to more recently developed small-molecule inhibitors. In this review, we will explore the molecular processes underlying the activation of several inflammasomes and highlight their role during health and disease. We also describe the detrimental effects of these inflammasome complexes, in some pathological conditions, and review current therapeutic approaches as well as future prospective treatments.

Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood-brain barrier in rodents and primates.

The development of gene therapies for the treatment of diseases of the central nervous system has been hindered by the limited availability of adeno-associated viruses (AAVs) that efficiently traverse the blood-brain barrier (BBB). Here, we report the rational design of AAV9 variants displaying cell-penetrating peptides on the viral capsid and the identification of two variants, AAV.CPP.16 and AAV.CPP.21, with improved transduction efficiencies of cells of the central nervous system on systemic delivery (6- to 249-fold across 4 mouse strains and 5-fold in cynomolgus macaques, with respect to the AAV9 parent vector). We also show that the neurotropism of AAV.CPP.16 is retained in young and adult macaques, that this variant displays enhanced transcytosis at the BBB as well as increased efficiency of cellular transduction relative to AAV9, and that it can be used to deliver antitumour payloads in a mouse model of glioblastoma. AAV capsids that can efficiently penetrate the BBB will facilitate the clinical translation of gene therapies aimed at the central nervous system.