Advancing AAV gene therapy for frontotemporal dementia


A recent publication by Sevigny (Eli Lilly and Company) et al. in Nature Medicine describes the development of a new AAV gene therapy for frontotemporal dementia. The publication outlines the program progression using several disease models and techniques from preclinical into clinical trials in a very nice way. This is a summary of the various models and techniques used in the drug discovery process.

AAV vector in frontotemporal dementia

Image is for illustrative purposes only and is not connected to the publication described.

The therapeutic gene and mechanisms of action

Frontotemporal dementia is a group of clinical syndromes characterized by progressive behaviorall, executive, linguistic, and motor dysfunction affecting the frontal and temporal lobes of the brain. There is a direct correlation between the levels of the protein progranulin and disease progression. A mutation in one of the two chromosomal alleles of the GRN gene, which codes for the progranulin protein, results in haploinsufficiency, reducing normal progranulin levels by approximately 50% and nearly guaranteeing the development of frontotemporal dementia.

The therapy aimed to restore progranulin levels in the brain and reduce lysosomal dysfunction, brain damage, and symptom progression using a non-replicating rAAV9 carrying a GRN cassette. The cassette contains the human GRN cDNA, engineered with optimized human codons, under the control of a CMVe and CBA promoter. The 3′ region also includes a woodchuck hepatitis virus post-transcriptional regulatory element, followed by a bovine growth hormone polyadenylation (bGH poly(A)) tail. The investigational gene therapy is called PR006.

CMVe promoter: Cytomegalovirus immediate-early enhancer, which enhances the activity of the promoter.
CBA promoter: Chicken β-actin promoter that ensures strong and consistent expression. Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE): Enhances the stability and translation of the produced mRNA.
Bovine growth hormone polyadenylation (bGH poly(A)) tail: A sequence that signals the end of
the transcription and adds stability to the mRNA.

Protein expression in iPSCs

Protein expression was analyzed using ELISA on iPSCs derived from patients with frontotemporal dementia. Cells from two patients carrying different mutations in the GRN gene were reprogrammed into proliferating neural stem cell lines, differentiated into neurons, and then transduced with PR006. The progranulin level post-treatment indicated successful gene delivery and expression in the cellular model.

neuron treated with AAV

Image is for illustrative purposes only and is not connected to the publication described.

Biodistribution and transgene expression in Grn-knockout mice

The vector’s biodistribution, the transgene’s expression, the activation of immune-related pathways, and histology were performed on Grn-knockout mice. Mice carrying a deletion in exons 1-4 of the Grn gene were injected with a single unilateral intracerebroventricular (ICV) injection of PR006 into the brain’s left hemisphere. After study termination, blood was collected, and brain tissue was divided into the hippocampus, motor cortex, somatosensory cortex, rest of the cortex, striatum, cerebellum, brainstem, and rest of the brain. Several other peripheral organs, including the gonads, kidneys, heart, liver, lungs, and spleen, were also collected.

Genomic DNA was extracted from the brain parts and peripheral tissues to investigate the biodistribution of the vector genome. The vector copy number was determined using qPCR and showed detectable levels of PR006 in the brain, indicating distribution and transduction efficiency.

Increased levels of GRN mRNA in the brains of Grn-KO mice after PR006 treatment indicate successful delivery and expression of the progranulin gene. The mRNA levels were quantified using RT-qPCR.

Note from us: The qPCR assay’s specificity depends mostly on how the primers and probes are designed. This can be challenging in samples with highly similar endogenous sequences. When the vector is the target, at least one of the primers should be directed toward the vector to exclude endogenous sequences. For the RNA transcript, transgene-specific sequences should be targeted; in this case, the sequence used optimized codons, probably differentiating it from the endogenous transcripts. In early model systems, the gene and its transcripts might be completely absent, but the assay should be designed and validated to transfer it to later models and clinical trials.

Measuring pathological features associated with frontotemporal dementia

Neuroinflammation is common in frontotemporal dementia associated with GRN mutations. To determine the effect of PR006 treatment on neuroinflammation, total RNA was extracted from the Grn-KO mice tissues, and the expression of the biomarkers TNF and CD68 was measured using RT-qPCR. The expression of both genes decreased after PR006 treatment, suggesting mitigation of neuroinflammation in these mice’s CNS.

RNA-seq was used to study the effect of PR006 treatment on lysosomal and immune-related pathways. Total RNA was extracted from fresh-frozen mouse cortex. PolyA enrichment was performed to isolate the mRNA. A cDNA library was prepared from the mRNA and analyzed using an Illumina HiSeq instrument. The mouse reference genome used for the analysis was modified to include the GRN transgene. Gene set variation analysis (GSVA) was performed to evaluate changes in specific gene sets related to lysosomes, the complement system, and vacuoles in the brain tissue. The activity levels were compared to previously published gene signatures dysregulated in Grn-KO mice. The activity state was then scored for the curated gene sets related to lysosomal and immune-related pathways to determine how active or inactive these pathways were. The results revealed that treatment with PR006 dose-dependently reversed the gene set deficiencies, particularly in genes associated with lysosomes, the complement system, and vacuole-related genes, indicating a restoration of gene expression patterns toward normal levels after PR006 treatment.

Histology and protein quantification in PR006-treated Grn-KO mice

Fixed sections of the brain were stained with hematoxylin and eosin, as well as immunostained for markers of neuroinflammation and neurodegenerative disease (Iba1; ionized calcium-binding adapter molecule 1) and an astrocyte marker (GFAP; glial fibrillary acidic protein). The results indicated potential alterations in neuroinflammatory processes and glial cell activation post-PR006 administration.

The quantitative data on progranulin production was obtained using ELISA, providing insights into the bioactivity of PR006 in transducing cells throughout the central nervous system (CNS). This supported the therapeutic potential of PR006 in upregulating progranulin levels in the brain tissue of Grn-KO animal models.

Toxicology in non-human primates (NHP)

The safety profile and potential adverse effects were studied in cynomolgus macaques before advancing to human trials. PR006 was administered once via ICM injection with a 6-d, 29-d or 182-d post-administration observation period and after trial ending tissues were harvested for histopathological investigations and to determining the gene expression levels using RT-qPCR.

PR006 was generally well tolerated, with minimal adverse effects. Minimal and asymptomatic dorsal root ganglionopathy was detected in the non-human primates (NHP) following PR006 administration suggests a localized neurological effect in the peripheral nervous system.

Despite the observed dorsal root ganglionopathy, the overall safety profile of PR006 in NHP was favorable, with no significant systemic toxicity or severe adverse events reported in the study.

Image is for illustrative purposes only and is not connected to the publication described.

The phase 1/2 clinical trial

This study (NCT04408625) was a Phase 1/2, multi-center, open-label, ascending-dose, first-in-human trial. It aims to evaluate the safety, tolerability, immunogenicity, effects on progranulin levels, biomarkers, and efficacy parameters of PR006.

The PR006 low dose was selected as the starting clinical dose. It corresponds to a dose with robust therapeutic benefits seen in animal models and maintains a reasonable margin to exposure to the non-adverse NHP toxicology findings. The study also contains mid-dose cohorts since the treatment was well tolerated by the participants. PR006 was administered as a single dose via suboccipital injection into the cisterna magna, a direct delivery into the CFS surrounding the brain and spinal cord.  All patients received immunosuppressant treatment. Titering methodology was updated during the study, and the present dose values are based on digital PCR methodology.

In total, 13 patients who met the enrollment criteria of symptomatic frontotemporal dementia and a progranulin mutation were included in the study. The study reported a total of 12 serious adverse events during the phase 1/2 trial. Among these, three patients developed deep vein thrombosis, a potentially life-threatening condition characterized by blood clot formation in deep veins.

Digital PCR (dPCR) in short: In digital PCR, the sample is partitioned into thousands of compartments, and an individual reaction is conducted in each compartment. Each compartment contains one or no target, allowing for direct counting of the target sequences in the sample in an absolute manner without the need for a standard curve. dPCR is less sensitive to PCR inhibitors and is the technique of choice for low-abundance targets in complex matrices, requiring high accuracy and precision.

The primary endpoint was changes in progranulin levels in CSF and blood, along with safety evaluations and immunogenicity assessments.

The progranulin protein was measured in human CSF and plasma using ELISA assays at various times, including before and at 2, 6, and 12 months post-treatment. An ELISA assay was also used as an anti-drug antibody assay against AAV9 capsid and progranulin to understand their impact on the efficacy of PR006 treatment.CFS progranulin increased post-treatment in all patients and blood granulin increased in most patients but only transient. 

Additionally, CSF pleocytosis, an increase in white blood cells in the CSF, was measured as a common PR006-related adverse event, reflecting potential immune responses to the gene therapy. In five of the six patients, the CSF pleocytosis was asymptomatic, indicating that the inflammatory response did not manifest clinically in most cases. However, one patient experienced decreased hearing as a symptom associated with the CSF pleocytosis, although this was reported to be recovering at the time of the interim analysis. The pleocytosis was detected in the cerebrospinal fluid (CSF) samples collected at month two post-treatment and was characterized by an increased number of white blood cells.

The secondary endpoint involved using the Clinical Dementia Rating (CDR) plus National Alzheimer’s Disease Coordinating Center (NACC) Frontotemporal Lobar Degeneration (FTLD) rating scale to measure cognitive and functional decline, as well as monitoring neurofilament light chain (NfL) levels as a biomarker for neurodegeneration.

The study evaluated the impact of PR006 on various domains affected in frontotemporal dementia, such as memory, orientation, judgment, behavior, and language, using the CDR plus NACC FTLD scale. In both the low-dose and mid-dose cohorts, the sum of boxes total scores increased, indicating a worsening of symptoms or disease progression at month six and month 12 post-PR006 gene therapy administration. The progression rates observed in the CDR plus NACC FTLD scale were within the broad ranges reported for patients with FTD, indicating typical disease progression in the study population.

To evaluate neurodegeneration and acute central nervous system inflammation post-administration, the neurofilament light chain (NfL) levels were measured using an immunoassay platform (Quanterix Simoa) in CSF and blood. In the study, measurements of NfL levels in blood showed a transient increase following PR006 treatment. The transient rise in blood NfL levels was likely attributed to the transduction and overexpression of the transgene in the dorsal root ganglia (DRG), leading to a secondary inflammatory response. Patients with a baseline blood anti-AAV9 titer tended to exhibit lower increases in plasma NfL levels, suggesting that pre-existing anti-AAV9 antibodies may have attenuated the transduction of PR006 in the DRG. The study did not report any signs or symptoms of sensory neuron dysfunction despite the transient increase in blood NfL, indicating that the observed changes in NfL levels were not associated with detectable neurological deficits.

The exploratory endpoint was to assess BMP, which is required for adequate lipid degradation by increasing the catabolic activities of lysosomal enzymes. Urinary BMP levels represent a potentially useful biomarker of lysosomal dysfunction. BMP levels were measured in human urine using LC-MS/MS as a downstream pharmacodynamic readout. BMP plays a crucial role in lysosomal function by enhancing the catabolic activities of lysosomal enzymes, which are essential for lipid degradation. Urinary levels of BMP serve as a biomarker reflective of lysosomal dysfunction and brain pathology, making it a valuable indicator for assessing the impact of PR006 gene therapy on lysosomal function in patients. The observed increases in urine levels of specific BMP species following PR006 treatment suggest a pharmacological effect of the gene therapy on lysosomal function, supporting its potential therapeutic efficacy in addressing the underlying pathophysiology.


All in all, this publication allows us to follow the entire drug discovery process through various therapeutic models from iPSC to first-in-human, and a toolbox of techniques including qPCR and RNA-seq to track the vector, transgene, and biomarker expression, as well as several methods for protein detection including LC-MS/MS, ELISA, and IHC. In addition to being a well-conducted study, nicely described in the publication, most importantly, the AAV gene therapy drug shows promising potential to help patients with frontotemporal dementia.

qPCR, dPCR, NGS and protein profiling for biopharma is what we do: If you are working in a drug development program and need method development, optimization, qualification, validation, and analysis of your qPCR, dPCR, or NGS assays, don’t hesitate to contact us at TATAA Biocenter. We are a GLP-accredited, GCLP, and ISO/IEC 17025-compliant laboratory specializing in nucleic acid analyses during the development of cell and gene therapies.


Sevigny, J., Uspenskaya, O., Heckman, L.D. et al. Progranulin AAV gene therapy for frontotemporal dementia: translational studies and phase 1/2 trial interim results. Nat Med (2024). https://doi.org/10.1038/s41591-024-02973-0

If you want to dig deeper into the results and look at the informative illustrations, the full article can be found here: https://www.nature.com/articles/s41591-024-02973-0

TATAA Biocenter has not been involved in generating any of the data included in the publication.