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

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 .

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice.

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this 'privileged' repertoire. Our experiments identify early transcription as a potential 'epigenetic' contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.

An Expression Refinement Process Ensures Singular Odorant Receptor Gene Choice.

Odorant receptor (OR) gene choice in mammals is a paradigmatic example of monogenic and monoallelic transcriptional selection, in which each olfactory sensory neuron (OSN) chooses to express one OR allele from over 1,000 encoded in the genome [1-3]. This process, critical for generation of the circuit from nose to brain [4-6], is thought to occur in two steps: a slow initial phase that randomly activates a single OR allele, followed by a rapid feedback that halts subsequent expression [7-14]. Inherent in this model is a finite failure rate wherein multiple OR alleles may be activated prior to feedback suppression [15, 16]. Confronted with more than one receptor, the neuron would need to activate a refinement mechanism to eliminate multigenic OR expression and resolve unique neuronal identity [16], critical to the generation of the circuit from nose to olfactory bulb. Here we used a genetic approach in mice to reveal a new facet of OR regulation that corrects adventitious activation of multiple OR alleles, restoring monogenic OR expression and unique neuronal identity. Using the tetM71tg model system, in which the M71 OR is expressed in >95% of mature OSNs and potently suppresses the expression of the endogenous OR repertoire [10], we provide clear evidence of a post-selection refinement (PSR) process that winnows down the number of ORs. We further demonstrate that PSR efficiency is linked to OR expression level, suggesting an underlying competitive process and shedding light on OR gene switching and the fundamental mechanism of singular OR choice.

Olfactory deficits cause anxiety-like behaviors in mice.

Anxiety disorders are characterized by persistent fear in the absence of immediate threat and represent the most common psychiatric diseases, with an estimated 28% lifetime prevalence worldwide (Kessler et al., 2010). While symptoms of anxiety are typically evoked by sensory stimuli, it is unknown whether sensory deficits contribute to the development of anxiety disorders. Here we examine the effect of defined genetic mutations that compromise the function of the olfactory system on the development of anxiety-like behaviors in mice. We show that the functional inactivation of the main olfactory epithelium, but not the vomeronasal organ, causes elevated levels of anxiety. Anxiety-like behaviors are also observed in mice with a monoclonal nose, that are able to detect and discriminate odors but in which the patterns of odor-evoked neural activity are perturbed. In these mice, plasma corticosterone levels are elevated, suggesting that olfactory deficits can lead to chronic stress. These results demonstrate a central role for olfactory sensory cues in modulating anxiety in mice.

Mice with a "monoclonal nose": perturbations in an olfactory map impair odor discrimination.

We have altered the neural representation of odors in the brain by generating a mouse with a "monoclonal nose" in which greater than 95% of the sensory neurons express a single odorant receptor, M71. As a consequence, the frequency of sensory neurons expressing endogenous receptor genes is reduced 20-fold. We observe that these mice can smell, but odor discrimination and performance in associative olfactory learning tasks are impaired. However, these mice cannot detect the M71 ligand acetophenone despite the observation that virtually all sensory neurons and glomeruli are activated by this odor. The M71 transgenic mice readily detect other odors in the presence of acetophenone. These observations have implications for how receptor activation in the periphery is represented in the brain and how these representations encode odors.

Regulation of odorant receptors: one allele at a time.

The odorant receptors (ORs) make up the largest gene family in mammals. Each olfactory sensory neuron chooses just one OR from the more than 1000 possibilities encoded in the genome and transcribes it from just one allele. This process generates great neuronal diversity and forms the basis for the development and logic of the olfactory circuit between the nose and the brain. The mechanism behind this monoallelic regulation has been the subject of intense speculation and increasing experimental investigation, yet remains enigmatic. Recent genetic experiments have brought the outlines of the process into sharper relief, identifying a feedback mechanism in which the first odorant receptor expressed, generates a signal that stabilizes its choice, thus maintaining singular selection. In the absence of this signal, the olfactory neuron re-enters the selection process and switches to choose an alternate OR. Irreversible genetic changes in the nuclei of olfactory neurons do not accompany OR selection, which must therefore be initiated by an epigenetic process that may involve a stochastic mechanism.

Gene switching and the stability of odorant receptor gene choice.

Individual olfactory sensory neurons express only a single odorant receptor from a large family of genes, and this singularity is an essential feature in models of olfactory perception. We have devised a genetic strategy to examine the stability of receptor choice. We observe that immature olfactory sensory neurons that express a given odorant receptor can switch receptor expression, albeit at low frequency. Neurons that express a mutant receptor gene switch receptor transcription with significantly greater probability, suggesting that the expression of a functional odorant receptor elicits a feedback signal that terminates switching. This process of receptor gene switching assures that a neuron will ultimately express a functional receptor and that the choice of this receptor will remain stable for the life of the cell.

Odorant receptor expressed sequence tags demonstrate olfactory expression of over 400 genes, extensive alternate splicing and unequal expression levels.

BACKGROUND: The olfactory receptor gene family is one of the largest in the mammalian genome. Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few olfactory receptors. We therefore screened a mouse olfactory epithelium cDNA library to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for many additional olfactory receptors, as well as identifying gene structure and putative promoter regions. RESULTS: We identified more than 1,200 odorant receptor cDNAs representing more than 400 genes. Using real-time PCR to confirm expression level differences suggested by our screen, we find that transcript levels in the olfactory epithelium can differ between olfactory receptors by up to 300-fold. Differences for one gene pair are apparently due to both unequal numbers of expressing cells and unequal transcript levels per expressing cell. At least two-thirds of olfactory receptors exhibit multiple transcriptional variants, with alternative isoforms of both 5' and 3' untranslated regions. Some transcripts (5%) utilize splice sites within the coding region, contrary to the stereotyped olfactory receptor gene structure. Most atypical transcripts encode nonfunctional olfactory receptors, but can occasionally increase receptor diversity. CONCLUSIONS: Our cDNA collection confirms olfactory function of over one-third of the intact mouse olfactory receptors. Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity. Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors. We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes.