Adeno-associated virus serotype 1-based gene therapy for FTD caused by GRN mutations

Abstract

Objective: Dominant loss-of-function mutations in the gene encoding the lysosomal protein, progranulin, cause 5-10% of frontotemporal dementia cases. As progranulin undergoes secretion and endocytosis, a small number of progranulin-expressing cells can potentially supply the protein to the entire central nervous system. Thus, gene therapy is a promising treatment approach.

Methods: We evaluated adeno-associated viral vector administration into the cerebrospinal fluid as a minimally invasive approach to deliver the granulin gene to the central nervous system in a murine disease model and nonhuman primates.

Results: In progranulin-deficient mice, vector delivery into the lateral cerebral ventricles increased progranulin levels in the cerebrospinal fluid and normalized histological and biochemical markers of progranulin deficiency. A single vector injection into the cisterna magna of nonhuman primates achieved CSF progranulin concentrations up to 40-fold higher than those of normal human subjects and exceeded CSF progranulin levels of successfully treated mice. Animals treated with an adeno-associated virus serotype 1 vector exhibited progranulin expression fivefold higher than those treated with an AAV5 vector or the AAV9 variant, AAVhu68, apparently due to remarkably efficient transduction of ependymal cells. Progranulin expression mediated by adeno-associated viral vectors was well tolerated in nonhuman primates with no evidence of dose-limiting toxicity, even at vector doses that induced supraphysiologic progranulin expression.

Interpretation: These findings support the development of AAV1-based gene therapy for frontotemporal dementia caused by progranulin deficiency.

Figure 1

AAV‐mediated PGRN expression corrects lysosomal pathology in brains of young adult GRN^−/− mice. GRN^−/− mice (KO) or GRN^+/+ (WT) controls were treated with a single ICV injection of vehicle (PBS) or an AAVhu68 vector expressing human PGRN (10¹¹ GC) at two months of age (N = 10 per group). Animals were sacrificed 60 days after injection, and human PGRN was measured in (A) CSF and (B) the frontal lobes of the brain by ELISA. C, We measured hexosaminidase activity in brain samples. Brain PGRN concentration and Hex activity were normalized to total protein. D, We imaged unstained brain sections for auto‐fluorescent material (lipofuscin) in hippocampus, thalamus, and frontal cortex. E‐G, A blinded reviewer quantified lipofuscin deposits in hippocampus, thalamus, and frontal cortex. Lipofuscin counts are expressed per high‐power field. N = 10 per group except KO + PBS hippocampus, which is N = 8. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001, one‐way ANOVA followed by Tukey’s multiple comparisons test. Scale bar = 250 µm (cortex and thalamus), 500 µm (hippocampus).

Figure 2

AAV‐mediated PGRN expression corrects brain microgliosis in aged GRN^−/− mice. GRN^−/− mice (KO) or GRN^+/+ (WT) controls were treated with a single ICV injection of vehicle (PBS) or an AAVhu68 vector expressing human PGRN (10¹¹ GCs) at seven months of age. A, Animals were sacrificed four months after injection, and brain sections were stained for CD68. B‐D, A blinded reviewer quantified CD68‐positive areas in images of hippocampus, thalamus, and frontal cortex using ImageJ software. Areas are expressed per high‐power field. **P < 0.005, ***P < 0.001, ****P < 0.0001, one‐way ANOVA followed by Tukey’s multiple comparisons test. Scale bar = 500 µm.

Figure 3

Human PGRN expression in the CSF of rhesus macaques following ICM AAV delivery. Adult rhesus macaques were administered AAV1, AAV5, or AAVhu68 vectors (3 × 10¹³ GC) expressing human PGRN from a chicken beta‐actin promoter by ICM injection on study day 0 (N = 2 per vector). Two additional macaques were administered an AAVhu68 vector expressing hPGRN from a ubiquitin C promoter (AAVhu68 V2). We performed the ICM injection under fluoroscopic guidance. A, After confirming needle placement by fluoroscopy and CSF return, (B) injection of contrast material demonstrated distribution within the cisterna magna. C, Displacement of the contrast was apparent during subsequent vector infusion. We measured human PGRN in CSF of (D) treated macaques and (E) healthy adult human subjects by ELISA. Dotted line = limit of quantification for hPGRN in CSF at a 1:5 dilution.

Figure 4

Immune response to human PGRN in vector‐treated NHPs. After administering AAV1, AAV5, or AAVhu68 vectors (3 × 10¹³ GC) expressing human PGRN from a chicken beta‐actin promoter or a ubiquitin C promoter (AAVhu68 V2) into the cisterna magna, we collected CSF on a weekly basis for chemistry and cytology analysis. A, Increased leukocyte counts (predominantly small lymphocytes) were evident in most animals. We measured antibody responses to human PGRN by ELISA in (B) CSF and (C) serum of animals treated with AAVhu68 or AAV1. We did not evaluate antibodies against human PGRN in serum or CSF samples from animals treated with AAV5.

Figure 5

Characterization of brain transduction following ICM administration of AAV1 and AAVhu68 vectors to NHPs. Adult rhesus macaques received 3 × 10¹³ GC of AAVhu68 (N = 2) or AAV1 (N = 2) vectors expressing GFP from a chicken beta‐actin promoter by ICM injection. Animals were necropsied 28 days after vector administration. A, We analyzed sections of five regions of the right hemisphere of the brain by (B, inset D) GFP immunohistochemistry or (C, inset E) immunofluorescence with staining for GFP (green) and 4′,6‐diamidino‐2‐phenylindole (DAPI; blue). Co‐staining with markers of specific cell types (NeuN, GFAP, and Olig2) allowed us to quantify transduced (C, E, F) neurons, (G) astrocytes, and (H) oligodendrocytes. I, We calculated the mean transduction of each cell type for all sampled brain regions. We evaluated ependymal cell transduction by performing immunohistochemistry in multiple regions of the lateral ventricle and fourth ventricle of animals treated with AAVhu68 (J) and animal RA1826, who was treated with AAV1 (K). Error bars = standard error of the mean (SEM) of the five sections. Scale bars = 5 mm (B, C), 200 µm (D, E). 100 µm (J, K), 50 µm (F, G), 10 µm (H).