CRISPR targeting of mmu-miR-21a through a single adeno-associated virus vector prolongs survival of glioblastoma-bearing mice

Sep 30, 2026

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Glioblastoma is the most aggressive central nervous system malignancy, with limited effective treatments and poor survival despite advances in surgery, radiotherapy, and temozolomide. MicroRNA-21 (miR-21) acts as an oncogene and drives GB progression, making miR-21(a) a therapeutic target. This study proposes a single adeno-associated virus (AAV) strategy to target mmu-miR-21a using SaCas9 guided by a single-guide RNA. AAV8-mediated delivery induces a genomic deletion that lowers brain mmu-miR-21a, reduces tumor growth, and improves overall survival in GB-bearing mice.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages19

Reading Time48 min

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Introduction

Glioblastoma and therapeutic need

miR-21(a) as a target in glioblastoma

CRISPR-Cas9 and AAV delivery rationale

Original Article CRISPR targeting of mmu-miR-21a through a single adeno-associated virus vector prolongs survival of glioblastoma-bearing mice Lisa Nieland, 1,2 Anne B. Vrijmoet, 1, 10 Isabelle W. Jetten, 1, 10 David Ruﬁno-Ramos,3,4,5 Alexandra J.E.M. de Reus, 1 Koen Breyne, 1 Benjamin P. Kleinstiver,3,4,5 Casey A. Maguire, 1,6,7 Marike L.D. Broekman, 1,2,8,9 Xandra O. Breakeﬁeld,1 and Erik R. Abels2 1Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA; 2Department of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands; 3Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; 4Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; 5Department of Pathology, Harvard Medical School, Boston, MA 02115, USA; 6Department of Neurology, Massachusetts General Hospital, Boston, MA 02115, USA; 7Harvard Medical School, Boston, MA 02116, USA; 8Department of Neurosurgery, Haaglanden Medical Center, 2512 VA The Hague, the Netherlands; 9Department of Neurosurgery, Leiden University Medical Center, 2300 RC Leiden, the Netherlands Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. miR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector inﬂuenced glioma development, resulting in beneﬁcial anti-tumor outcomes in GB-bearing mice. INTRODUCTION Glioblastoma (GB) is the most common and aggressive form of malignant primary brain tumor of the central nervous system (CNS) .1 Despite advances in local therapy,2 the complex heterogeneity results in therapeutic resistance, high recurrence rates, and poor median survival outcomes of 􀀁 15 months.2,3 The 5-year overall survival rate is less than 7% with an estimated annual mortality of over 10,000 individuals in the United States.4 The current standard of care involves surgical resection followed by radiation therapy and temozolomide; this treatment standard has not changed over the past two decades.3 Therefore, the development of improved treatment strategies is urgently needed. The expression of microRNA-21 (miR-21) (human hsa-miR-21 ormurine mmu-miR-21a) is increased in nearly all cancers, including GB.5,6 miR-21 was ﬁrst discovered to be increased in human GB by Chan et al.,7 and since then many other studies have reported upregulated miR-21 levels in patient-derived glioma tumor samples and cerebrospinal ﬂuid from patients.7–9 Elevated miR-21 levels have been negatively correlated with malignancy and patients with increased miR-21 levels have a worse prognosis.10 Over the past years, miR-21(a) has therefore gained interest as a potential therapeutic target in many cancers, including GB.11 Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) have had a major impact in the ﬁeld of gene therapy in the last decade.12 Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing.13 Various delivery methods for Cas nucleases have been developed for cultured cells, including plasmid and RNA transfections. Additionally, viral-based delivery methods, including lentivirus, adenovi

FAQs

Why is miR-21(a) targeted in glioblastoma?

miR-21(a) is an oncogene whose increased expression promotes glioblastoma progression, and reducing miR-21(a) lowers tumorigenic potential.

What strategy does the study use to disrupt mmu-miR-21a?

It uses a single adeno-associated virus vector to deliver SaCas9 guided by a single-guide RNA (sgRNA) to target mmu-miR-21a.

How does the AAV-delivered SaCas9 affect tumor outcomes?

SaCas9 induces a genomic deletion that lowers mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival in glioblastoma-bearing mice.

Sep 30, 2026

Upload by Jiven