Progranulin AAV gene therapy for frontotemporal dementia: translational studies and phase 1/2 trial interim results

Abstract

GRN mutations cause progranulin haploinsufficiency, which eventually leads to frontotemporal dementia (FTD-GRN). PR006 is an investigational gene therapy delivering the granulin gene (GRN) using an adeno-associated virus serotype 9 (AAV9) vector. In non-clinical studies, PR006 transduced neurons derived from induced pluripotent stem cells of patients with FTD-GRN, resulted in progranulin expression and improvement of lipofuscin, lysosomal and neuroinflammation pathologies in Grn-knockout mice, and was well tolerated except for minimal, asymptomatic dorsal root ganglionopathy in non-human primates. We initiated a first-in-human phase 1/2 open-label trial. Here we report results of a pre-specified interim analysis triggered with the last treated patient of the low-dose cohort (n = 6) reaching the 12-month follow-up timepoint. We also include preliminary data from the mid-dose cohort (n = 7). Primary endpoints were safety, immunogenicity and change in progranulin levels in cerebrospinal fluid (CSF) and blood. Secondary endpoints were Clinical Dementia Rating (CDR) plus National Alzheimer's Disease Coordinating Center (NACC) Frontotemporal Lobar Degeneration (FTLD) rating scale and levels of neurofilament light chain (NfL). One-time administration of PR006 into the cisterna magna was generally safe and well tolerated. All patients developed treatment-emergent anti-AAV9 antibodies in the CSF, but none developed anti-progranulin antibodies. CSF pleocytosis was the most common PR006-related adverse event. Twelve serious adverse events occurred, mostly unrelated to PR006. Deep vein thrombosis developed in three patients. There was one death (unrelated) occurring 18 months after treatment. CSF progranulin increased after PR006 treatment in all patients; blood progranulin increased in most patients but only transiently. NfL levels transiently increased after PR006 treatment, likely reflecting dorsal root ganglia toxicity. Progression rates, based on the CDR scale, were within the broad ranges reported for patients with FTD. These data provide preliminary insights into the safety and bioactivity of PR006. Longer follow-up and additional studies are needed to confirm the safety and potential efficacy of PR006. ClinicalTrials.gov identifier: NCT04408625 .

Fig. 1. ICV delivery of PR006 in Grn-KO mice transduces cells throughout the CNS, increasing progranulin expression and reducing known pathologies.

Four-month-old Grn-KO mice were dosed with PR006 or excipient by ICV administration (n = 10, four males and six females per group in all assays (a–l)). Three months after dosing, animals were euthanized. a, Presence of vector genome per microgram of gDNA on a log scale. Dashed line (50 vg μg⁻¹ gDNA) represents the lower limit of detection. b, GRN RNA expression was assessed by qRT–PCR in the cerebral cortex. The number of GRN copies (specific to our codon-optimized PR006 sequence) was normalized to 1 µg of total RNA and is shown on a log scale. c, Progranulin protein levels were measured in the cortex and normalized to total protein concentration. d, Lipofuscin accumulation was semi-quantitatively scored in H&E-stained sections in the hippocampus. e, Immunohistochemical (IHC) analysis of ubiquitin was quantified in the hippocampus. The size of above-threshold immunoreactive objects was measured. f,g, mRNA levels of Tnf (f) and Cd68 (g) were measured by qRT–PCR in the cortex. h,i, IHC analysis of Iba1 (h) and GFAP (i) was quantified in fixed brain sections in the hippocampus. The percent of the area of interest (immunoreactive area) is shown (all data in a–i represented as mean ± s.e.m.). j–l, RNA sequencing in cerebral cortex samples is shown as the GSVA activity scores for curated gene sets Lysosome (j), Complement System (Hallmark pathway) (k) and Cellular Component: Vacuole (GO:0005773) (l) (data shown as box plots defined by the minimum, 1st quartile, 2nd quartile (median), 3rd quartile and the maximum). Statistical analysis was conducted using ANOVA followed by Dunnett’s test to compare to the excipient-treated Grn-KO mouse group, which kept the family-wise type I error rate at 0.05 rather than 0.15 as implied by the number of comparisons. Exact P values are reported in the figure where appropriate, accompanied by **P < 0.01, ***P < 0.001 and ****P < 0.0001. In d, excipient versus WT (P < 0.0001), versus 1.6 × 10¹⁰ (P = 0.0097) and versus 1.6 × 10¹¹ (P < 0.0001) were significantly different.

Fig. 2. Patient disposition.

Enrolled patients were 55–75 years old, had a confirmed pathogenic GRN mutation and had symptomatic FTD-GRN with CDR plus NACC FTLD sum of boxes score between 1 and 15. One patient was enrolled and remained in active screening at the time of submission for publication.

Fig. 3. PR006 increases progranulin levels in CSF of FTD-GRN study participants.

CSF samples were collected from study participants in the low-dose (2.1 × 10¹³ vg, n = 5) and mid-dose (4.2 × 10¹³ vg, n = 6) cohorts. Progranulin concentrations (ng ml⁻¹) in CSF samples were measured by ELISA and plotted against timepoint of collection (baseline and months 2, 6 and 12; additional timepoints were collected at months 3 and 4 for two study participants). At baseline, progranulin concentrations were all below normal levels, as reported by Goossens et al. (denoted in the figure by the red line). Upon dosing with PR006, at the month 2 timepoint, levels of progranulin concentration increased by 2.1–6.9× above the respective baseline levels. Among the nine patients with CSF progranulin data at month 6, eight (89%) had progranulin level within or above the normal range, whereas, among the four patients with 12-month data, three (75%) had levels within or above the normal range, as described by Goossens et al..

Fig. 4. Levels of total di-18:1-BMP and di-22:6-BMP in urine were increased upon PR006 dosing of patients with FTD-GRN.

Urine samples were collected from study participants in the low-dose (2.1 × 10¹³ vg, n = 6) and mid-dose (4.2 × 10¹³ vg, n = 6) cohorts. a,b, Total di-18:1-BMP (a) and di-22:6-BMP (b) levels were measured using a mass spectrometry–based assay and were normalized with creatinine concentrations (ng mg⁻¹). Mean (±s.e.m.) values were plotted against timepoints of sample collection (baseline and months 1, 2, 3, 6, 9 and 12). Percent of total di-18:1-BMP and di-22:6-BMP levels relative to baseline is shown. For both BMP species, an increase of BMP levels compared to baseline was observed. The increase of BMP was sustained until month 12 for the low-dose cohort and until month 3 for the mid-dose cohort. Data plots of individual patients are provided in Extended Data Fig. 6. The increased BMP levels approached values measured in urine samples obtained from healthy human controls, which were measured as 40.8 ± 8.4 and 50.4 ± 7.7, respectively, for di-18:1-BMP and di-22:6-BMP (mean ± s.e.m., n = 24).

Extended Data Fig. 1. PR006 transduces FTD-GRN mutation carrier neuronal cultures in a dose-dependent manner.

(a) PR006 consists of a non-replicating recombinant AAV of 4184 nucleotides which includes an expression cassette containing the codon optimized coding sequence of human Progranulin GRN. The viral particle (AAV capsid) is comprised of three capsid proteins, VP1, VP2, and VP3, with the theoretical molecular weights of approximately 87, 73, and 62 kDa. PR006 encapsidates the modified viral vector, including the GRN expression cassette. The vector contains flanking ITRs on each side of the expression cassette, the Cytomegalovirus enhancer (CMVe) and Chicken Beta Actin promoter (CBAp) to constitutively express the codon optimized coding sequence of human GRN. The 3′ region also contains a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) element followed by a bovine growth hormone polyadenylation (bGH polyA) tail. Further information is provided in US Patent 10689625, which covers composition claims for PR006 vector and rAAV. (b) Neural stem cells (NSCs) were seeded at an equal density and differentiated into neurons. On day 7, neurons were transduced with excipient or the indicated amounts of PR006 for 72 hours. Secreted progranulin expression was measured from the cell media by ELISA and normalized to volume (n=3–4; mean ± SEM). Black dashed line represents endogenous levels of secreted progranulin from Control neurons (excipient-treated). Secreted progranulin was not detectable in excipient-treated FTD-GRN neurons. Statistics were determined using ANOVA followed by Tukey HSD and statistical comparison of each condition to excipient-treated Control neurons is indicated on the graph. MOI, multiplicity of infection.

Extended Data Fig. 2. Reduced lysosomal and neuropathology defects in an aged FTD-GRN mouse model following PR006 treatment.

Tissue samples were collected from 18-month-old Grn-KO mice 2 months after receiving ICV excipient (n=4, 2 males and 2 females) or 5.8 x 10¹⁰ vg (1.5 x 10¹¹ vg/g brain) PR006 (n=5, 3 males and 2 females). (a) Representative lipofuscin images from 3 independent studies of the thalamus/hypothalamus region of brain sections (Left = Grn KO + Excipient; Right = Grn KO + PR006). White arrowheads indicate examples of lipofuscin accumulation. (b) A summary of lipofuscin severity scores in the cerebral cortex, hippocampus, and thalamus/hypothalamus of H&E-stained slides from brain sections that were evaluated for autofluorescent lipofuscin granules. Lipofuscin accumulation was semi-quantitatively scored by a blinded board-certified pathologist. (c) Urine samples were collected from Grn-KO mice 14 weeks after receiving ICV excipient or PR006 (n=9–10 per treatment group, 4–5 males and 4–5 females), while control (wild-type) urine (age, gender and strain matched) were purchased from BioIVT. BMPs were extracted by liquid-liquid extraction from all samples at the same time. BMP concentrations were measured using a SCIEX X500 LC-MS/MS system. Standard curves for di-22:6-BMP and di-18:1-BMP were prepared from corresponding authentic reference standards. Data show as boxplots defined by the minimum, 1^st quartile, 2^nd quartile (median), 3^rd quartile, and the maximum. Statistical analyses were performed using a Dunnett’s Test (a two-sided post-hoc test). Exact 0.05 < p < 0.0001 are reported in the figure where appropriate, accompanied by * = p < 0.05, ** = p < 0.01, *** = p< 0.001, ****p < 0.0001. In (b), excipient vs. PR006 was significant in Cerebral Cortex (p = 0.005), Hippocampus (p < 0.0001), and the Thalamus/Hypothalamus (p = 0.038).

Extended Data Fig. 3. Expression of PR006 transgene (GRN) in non-human primates 6 months post dosing.

PR006 was administered once via ICM injection in cynomolgus monkeys (n=3 per treatment group, 2 males and 1 female). Animals were sacrificed at study Day 183. The study evaluated 2 dose levels: a low dose of 2.9 × 10¹¹ vg and a high dose of 2.9 × 10¹² vg; with a brain weight estimate of 74 g in a cynomolgus monkey, the NHP species used in this study, this translates to approximately 3.9 × 10⁹ vg/g brain and 3.9 × 10¹⁰ vg/g brain. The study also included a control arm in which animals received 1.2 mL of excipient only. (a) Tissue samples were collected for qPCR analysis. GRN expression levels were determined in NHP cortex, hippocampus and ventral mesencephalon collected on Day 183 using RT-qPCR with primers designed for the PR006 human transgene. Data is presented as mean ± SEM. (b) Progranulin levels were measured in CSF samples from NHPs treated with excipient, low dose, or high dose of PR006 collected at Day 183. Progranulin levels were measured using a Simple Western (Jess) analysis. Data is presented as mean ± SEM; P-value: *p = .023, by one-way dose dependence response analysis using William’s trend test.

Extended Data Fig. 4. PR006 administration leads to transient elevations in plasma progranulin levels of FTD_GRN study participants without pre-existing anti-AAV9 antibodies.

Plasma samples were collected from study participants in the low-dose (2.1 × 10¹³ vg, n=5) and mid-dose 4.2 × 10¹³ vg, n=6) cohorts. Progranulin concentrations (ng/mL) in plasma samples were measured by ELISA and plotted against timepoint of collection (baseline, months 1, 2, 3, 6, 9, and 12). At baseline, progranulin concentrations were all below the cut-off value reported by Swift et al., denoted in figure by red line). Upon dosing with PR006, at the month 1 timepoint, levels of progranulin concentration increased by 12x to 36x above the respective baseline levels in 8 subjects, which tested negative for anti-AAV9 antibodies at baseline. In the 8 patients the levels generally decreased to baseline within 2–3 months (values not available for 1 subject). Five subjects tested positive for anti-AAV9 antibodies at baseline and showed minimal changes from baseline (0.8x to 1.8x).

Extended Data Fig. 5. PR006 administration leads to transient elevations in plasma and CSF NfL levels.

Plasma and CSF samples were collected from study participants in the low-dose (2.1 × 10¹³ vg, n=5) and mid-dose 4.2 × 10¹³ vg, n=6) cohorts. (a) NfL concentrations (pg/mL) in plasma samples were measured using the Quanterix Simoa NfL assay and plotted against timepoint of collection (baseline, months 1, 2, 3, 6, 9, and 12). Upon dosing with PR006, plasma levels increased by up to 11x above the respective baseline levels at 2 months and returned to baseline levels or slight elevations at 9–12 months after dosing. Elevations tended to be lower in subjects who tested positive for anti-AAV9 antibodies at baseline. (b) NfL concentrations (pg/mL) in the CSF were measured at baseline, and months 2, 6 and 12. Upon dosing with PR006, CSF levels increased up to 11x at 2 months and returned to baseline levels, reductions from baseline or slight elevations at 6 to 12 months after dosing.

Extended Data Fig. 6. Individual subject adta plots of di-18:1 and di-22:6-BMP in urine following PR006 dosing of FTD-GRN patients.

Urine samples were collected from study participants in the low dose (2.1 × 10¹³ vg, n=5) and mid dose 4.2 × 10¹³ vg, n=6) cohorts. Total (a = low dose, b = mid dose) di-18:1- and (c = low dose, d = mid dose) di-22:6-BMP levels were measured using a mass spectrometry-based assay and were normalized with creatinine concentrations (ng/mg). Mean (±SEM) values and statistical analyses are shown in Fig. 4 of the main text.