Aims

Aβ immunotherapy is seen as the first clinical therapy for AD that can alter underlying disease mechanisms. However, only modest cognitive benefits and potentially serious side effects limit effectiveness. Given continued poor understanding of the mechanisms by which Aβ immunotherapy work in the AD brain should lead to renewed experimental efforts. Although microglial-induced plaque removal is involved, our and increasingly other groups have over the years shown that damage from aggregating Aβ initiates within synaptic endosomes even prior to the appearance of amyloid plaques and in Tampellini D et al., 2017, we had reported that Aβ antibodies can be internalized, reduce intraneuronal Aβ and normalize synaptic alterations in AD transgenic neurons in culture. Here we will further define cellular mechanisms involved.

Methods

Prion-like N2a cells that maintain Aβ aggregates (Olsson TT et al., 2018; Roos TT et al., 2021), and primary mixed brain cultures and brains of AD model and WT mice are examined after antibody treatment. Unlabeled and fluorescently tagged Aβ antibodies are used. The endosome.lysosome-autophagy system is manipulated and examined by fluorescence microscopy.

Results

In preliminary results we confirm that Aβ antibodies are internalized into cells and in prion-like N2a cells with Aβ aggregates, they colocalize with the Aβ deposits. We are defining (markers and functionality) of the organelles with the Aβ antibodies/aggregates, which are Lamp1 positive.

Conclusions

The endosomal accumulation of Aβ near synapses initiates a biological process leading to synaptic dysfunction and eventual plaque formation and AD. We believe that more specifically targeting this initial aberrant Aβ accumulation can improve on Aβ immunotherapy for AD.

Aims

The major question addressed in this presentation is whether short-hairpin RNA (shRNA) molecules designed to target and knock down the human HTR2A gene can reversibly modify neuronal circuits and subsequently impact specific behaviors associated with anxiety and memory impairments.

Methods

The mouse HTR2A gene encodes a single protein-coding transcript, Htr2A-201 located on chromosome 14. To test shRNA targeted to knock down the HTR2A gene in vivo, AAV9-shRNA-designed plasmids were administered intranasally on day 1, and molecular as well as behavioral endpoints were measured 5 weeks later. Subcloning of the shRNA was carried out in a modified pAAV cis-plasmid under the neuronal-specific promoter, MeCP2. A reporter gene-enhanced green fluorescent protein (eGFP) was subcloned upstream of the shRNA sequence.

Results

•Evidence that in vitro, human iPSC-differentiated neurons treated with AAV9-HTR2A-shRNA constructed DNA plasmids leads to downregulation of 5HT-2A receptor mRNA, protein receptor density, and decreased electrical excitability following MEA analysis.

•Evidence that in vivo, intranasally administered AAV9-HTR2A-shRNA bypasses the blood-brain barrier and noninvasively downregulates 5HT-2A receptors leading to a significant decrease in anxiety behavior.

•Evidence that in vivo, intranasally administered AAV9-HTR2A-shRNA led to significant memory enhancements of up to 104% via intranasal delivery of AAV9 DNA plasmids. This approach, validated in both mice and aged rats, leverages two cutting-edge gene therapies (CRISPR/Cas9 and shRNA)—a compelling advancement with profound implications for the treatment of various neurological disorders including MCI, anxiety, and AD.

Conclusions

Our demonstration of a non-invasive shRNA delivery platform that can bypass the blood-brain barrier has broad implications for treating numerous neurological disorders. Specifically, targeting the HTR2A gene presents a novel therapeutic approach for treating anxiety and memory impairments associated with Alzheimer's disease.

Aims

Adeno-associated (AAV) vector is a platform-of-choice for delivery of genetic cargoes; nevertheless, its small packaging capacity mainly is not suitable for the delivery of bulky and complex CRISPRi tools. To circumvent this limitation, here we aimed to develop a compact dCas9-repressor system packaged within a single, optimized AAV backbone. We thought to validate the developed system on its ability to efficiency silence genetic targets in vitro and in vivo.

Methods

To circumvent a small packaging capacity of adeno-associated vectors, here we applied a robust reporter-based, single- virion’s resolution screening procedure to devise and select for a compact dCas9-based repressor system. The developed AAV-expression cassette has been successfully packaged within a single, optimized viral vector. The developed system uses a smaller dCas9 variant derived from Staphylococcus aureus (Sa). A novel repressor was engineered by fusing a small transcription repression domain (TRD) from MeCP2 protein with KRAB repression peptide. The final dSaCas9-KRAB-MeCP2(TRD) construct can be efficiently packaged, along with a gRNA, into AAV particles.

Results

The AAV-based system, outlined in this study has been found to be efficient and robust for inducing a long-term and sustainably repression of the expression of GFP/Luciferase reporters, as well as for downregulating expression of ApoE gene, the primary genetic risk factor for late onset Alzheimer’s disease (LOAD).

Conclusions

Here, we developed a compact dCas9-repressor system packaged within a single, optimized AAV vector. This innovative viral platform will broaden CRISPRi toolsets utilized for transcriptional manipulation of gene expression in research and therapeutic settings.

Aims

Evidence of early damage to the locus coeruleus (LC) in neurodegenerative disorders suggests that a noradrenergic deficit may underpin decline in these diseases. This trial explored whether cognitive deficits are rescued by treatment with the CNS-penetrant, oral β₂-AR agonist, CST-2032, in patients with cognitive impairment due to AD or PD.

Methods

Blinded oral CST-2032 (3mg) + nadolol (3mg), or placebo was administered for 2 weeks in a 2-period crossover design to patients with MCI or mild dementia due to PD or probable AD, and Montreal Cognitive Assessment (MoCA) score between 14-26. Measures of cognition included the digit symbol substitution test (DSST), Cambridge neuropsychological test battery (CANTAB), and the facial expression recognition task (FERT).

CST-2032 was administered with a low dose of nadolol, a β₂-AR preferring antagonist with negligible CNS absorption, to inhibit peripheral effects of β₂-AR agonism.

Results

64 patients were randomized, 36 with probable AD and 28 with PD, with an average age of 68 years and MoCA score of 21.1 at enrollment.

At present, data are available only for participants with probable AD in whom significant improvements in cognition were observed with CST-2032+nadolol in patient subgroups including significant increases in the DSST score in subjects with MCI_AD (LS mean difference from placebo=1.88 [p=0.033] on Day 14) with concomitant reductions in reaction times for FERT tasks. The PD patient cohort is ongoing and expected to complete before year end.

CST-2032+nadolol had an excellent safety and tolerability profile. Most treatment related AEs were mild and there were no SAEs related to treatment.

Conclusions

The β₂-AR agonist, CST-2032+nadolol improved performance in cognitive tasks and was well-tolerated. These data support longer-term evaluation of CST-2032+nadolol.

Aims

Therapeutics that preserve synapses are an important goal towards treating neurodegenerative diseases. Synapse toxicity in AD and PD , are driven by amyloid-β oligomers (Aβo) and α-synuclein oligomers (αSo) respectively, binding to the cellular prion protein (PrP^C) and metabotropic glutamate receptor subtype 5 (mGluR5) receptor complex. We have identified ALX-001 (formerly BMS-984923) a first-in-class orally bioavailable silent allosteric modulator of mGluR5 which selectively blocks only Aβo and αSo signaling and preserves normal glutamate function. The aim of this study was a Phase 1b clinical trial to evalaute the safety, tolerability, and pharmacokinetics of ALX-001 with twice daily oral administration for up to 20-days in healthy older adults.

Methods

Twenty-four participants between the ages of 50 to 80 years old with normal cognition were enrolled in a placebo controlled, double-blinded, mutiple ascending dose study. Four cohorts of eight participants each, randomized 6:2 drug to placebo, were administered ALX-001 twice daily for either ten or twenty days. Safety evaluations and plasma collections were conducted regularly. Safety reviews were conducted prior to escalation to the next cohort.

Results

All doses of ALX-001 were well tolerated without serious adverse events. All treatment emergent adverse events (TEAEs) were mild in intensity and only 19 TEAEs were considered possibly related to treatment. Plasma pharmacokinetic properties were characterized for each dose, demonstrating successful acheivement of steady state by day 20.

Conclusions

ALX-001 is safe and well tolerated for 20-days with twice daily oral dosing. Plasma exposures reached levels achieve full coverage of the IC₈₀ for receptor occupancy at trough which is expected to yield therapeutic benefit in patients. Further studies are planned to assess the safety, tolerability, and potential efficacy of ALX-001 in patients with AD and PD.

Aims

Identification and validation of septins as a novel drug target for neuroprotection and functional restoration of neural circuits in Alzheimer’s disease (AD).

Methods

Small molecule phenotypic screen in cell-based model of tau-driven toxicity. Target deconvolution using a 3-hybrid screen and cell-free binding studies. Molecular dynamics simulations. Efficacy studies in patient iPSC-derived cortical neurons and AD mouse models. Randomized, double-blind, placebo controlled Ph2a study in mild to moderate AD patients.

Results

A small-molecule screen in a tau-based cell model of neurotoxicity led to the identification of REM0046127 as a potent neuroprotective agent. Efficacy studies in both APP- and tau-based mouse models of neurodegeneration showed that REM0046127 restores synaptic plasticity (LTP), normalizes network oscillatory activity (EEG) and rescues spatial memory. In addition, REM0046127 reduces amyloid β plaque load and tau hyperphosphorylation. Target deconvolution identified septins (with highest binding affinity to septin 6) as the functional target of REM0046127. We show that pathogenic tau destabilizes the septin cytoskeleton causing abnormal activation of store-operated calcium channels (SOCCs) and calcium overload in the cytoplasm. Binding of REM0046127 to septins restores integrity of septin filaments and restrains calcium entry through SOCCs. Preliminary Phase 2a data of AD patients revealed that REM0046127 improved cognition, normalized brain rhythms and restored CSF levels of the neurotransmitters acetylcholine and dopamine. The compound also impacted amyloid β and tau CSF biomarkers consistent with its effect on cognition.

Conclusions

Our findings provide support for calcium-driven neurodegeneration and identify the septin cytoskeleton as a novel and promising therapeutic target in AD. Modulation of septin assembly by REM0046127 results in symptomatic relief and disease modification in AD preclinical models. Preliminary data from the ongoing Phase 2a study suggest these findings may translate to AD patients.

Aims

A Phase 2 single-arm study of ALZ-801/valiltramiprosate (NCT04693520) evaluated plasma p-tau as primary outcome (Abushakra CTAD 2022). We compared ALZ-801 effects on hippocampal volume (HV) and cognitive outcomes to an external-control arm from the AD Neuroimaging Initiative (ADNI-1). We present the regulatory basis for using external controls and the statistical methods that minimize potential bias from this approach.

Methods

The US FDA recently provided a regulatory framework to advance use of real-world evidence to support drug development (RWE Guidance, 2023), addressing use of external controls for single-arm trials. Potential biases in such comparisons stem from differences in ethnicity/geographies, medical comorbidities, and changes in diagnostic-criteria and standard-of-care treatments. FDA recommends using the study’s inclusion/exclusion (I/E) criteria to select subjects from a non-interventional study, followed by subject-level matching for important variables that impact disease trajectory, and use of propensity-score for more robust matching. Statistical analyses include these matched variables as covariates in ANCOVA models.

Results

Using I/E of the Phase 2 study, 151 ADNI-1 subjects were selected who are APOE4 carriers with Early AD, aged 50-80 years. From these 151, 82 subjects matched 79 subjects from the ALZ-801 study. For overall and subject-matched groups, ANCOVA analyses including MMRM showed significant benefits of ALZ-801 for the HV and cognitive tests at 104 weeks (Hey, ADPD 2024). Results of propensity-score matching will be presented.

Conclusions

Applying established RWE guidance, we compared ALZ-801 Phase 2 data to a well-matched ADNI-1 cohort while adjusting for their baseline differences. The consistent and large effects of ALZ-801 on hippocampal volume and cognitive measures compared to the ADNI-control, across various analytical methods, support the efficacy of ALZ-801 in APOE4 carriers with Early AD and its disease modifying profile.

Aims

CSF proteomics was used as an exploratory biomarker to assess effects of semorinemab in the Tauriel Study (NCT03289143) and the Lauriet Study (NCT03828747) which are Phase 2 randomized, double-blind, placebo-controlled, parallel-group clinical trials that assessed the safety and efficacy of semorinemab in patients with prodromal to moderate Alzheimer’s disease combined.

Methods

CSF was collected from a subset of patients at baseline and after 49 or 73 weeks in Tauriel and baseline and after 49 or 61 weeks for Lauriet. Samples were analyzed using single-shot FAIMS-DIA-MS and analyzed using Spectronaut and MS Stats and normalized to global median intensity.

Results

"Drug signatures'' were defined for each trial as the set of genes encoding proteins which were elevated under semorinemab treatment. Based on single-cell RNAseq data from 3 independent studies obtained from brain samples of AD and healthy donors, Lauriet drug signature genes were robustly enriched in microglia. Furthermore, the Lauriet drug signature was significantly upregulated in microglia from AD patients vs. non-demented controls. Additionally, in Lauriet, CHI3L1 and GPNMB proteins were increased with treatment, suggestive of an activated microglia state.

Conclusions

Analysis of the Lauriet and Tauriel CSF signature indicate that the Lauriet proteomic drug response are likely of microglia origin, including some contribution from proteins that are further elevated in Alzheimer’s-associated microglia.