PACS2 Cure Roadmap
The PACS2 Research Foundation commissioned the first version of what will be an evolving strategic plan that leads to new discoveries and responds to translational opportunities as they arise.
PACS2 Syndrome Cure Roadmap
Prepared for PACS2 Research Foundation by Perlara PBC
Toward a future where all people with PACS2 syndrome live healthy, fulfilled, and complete lives. This multi-year, multi-modality roadmap presents scientific and therapeutic strategies to find a treatment for patients with PACS2 syndrome. Based on the current state of knowledge on PACS2 syndrome, we recommend the development and characterization of several cellular disease models and pursuing drug repurposing and antisense oligonucleotide therapies for this condition.
The goal of the PACS2 syndrome Cure Roadmap is to stimulate the development of medicines that will treat PACS2 syndrome:
Our key recommendations for the next 12-18 months are:
● Cellular Models: Patient-derived fibroblasts: In collaboration with Charles River Laboratories, characterize Lena’s fibroblast cells by Cell Painting to identify screenable phenotypes, and develop an assay to enable high-throughput screening of repurposed drugs.
● Cellular Models: iPSC-neurons: collaborate with Dr. Guemez-Gamboa to generate and phenotype iPSC-neuron models of PACS2 syndrome. The cell lines can be employed in several therapeutic tracks to validate therapeutic candidates in a disease-relevant cellular model.
● Therapeutic Track 1: Drug repurposing (in vitro): focus on phenotypic-based drug repurposing screen in PACS2 syndrome patient-derived fibroblasts using the Broad Repurposing Hub compound library in collaboration with Charles River Laboratories.
● Therapeutic Track 2: ASO precision medicine: design and screen allele-specific antisense oligonucleotides by the n-Lorem Foundation program.
● Therapeutic Track 3: PROTACs: explore targeted protein degradation as a therapeutic avenue for PACS2 syndrome by engaging in conversation with key opinion leaders and biotechnology companies.
Kristin Kantautas, PhD (Cure Guide Perlara, PBC)
Ethan O. Perlstein, PhD (CEO of Perlara PBC & Maggie’s Pearl LLC)
We invite a post-publication review of this Cure Roadmap here on our Substack.
Lena is a 1-year girl who was born with PACS2 syndrome, an ultra-rare neurodevelopmental disorder caused by a de novo missense mutation c.625G>A (p.Glu209Lys) in the PACS2 gene. PACS2 syndrome is a developmental and epileptic encephalopathy (DEE) characterized by early-onset epilepsy, global developmental delay with hypotonia, intellectual disability, mild to severe cerebellar dysgenesis and facial dysmorphisms (Dentici et al., 2019; Mizuno et al., 2021; Olson et al., 2018). Some individuals with PACS2 syndrome may present with autism (Olson et al., 2018). Since mutations in the PACS2 gene were associated with a DEE in 2018, fewer than 20 patients have been reported in the literature. Interestingly, almost all reported PACS2 syndrome patients carry the same E209K pathogenic variant. A single case of a maternally inherited E209K has been reported, as well as one case of a de novo c.631G>A (p.Glu211Lys) variant (Cesaroni et al., 2022). Following Lena’s diagnosis, her parents launched PACS2 Research Foundation to discover a treatment for Lena and all children with PACS2 syndrome. Nearly 50 patients have been diagnosed with PACS2 syndrome worldwide. The true prevalence and incidence of PACS2 syndrome are still unknown.
The PACS2 (phosphofurin acid cluster sorting protein 2) gene is located on Chromosome 14q32.33 and contains 25 exons and 20 alternatively spliced variants (Ensembl). The recurrent c.625G>A mutation is located in exon 6 which is present in all PACS2 protein-coding transcripts (Ensembl). The PACS2 gene is broadly expressed in the brain with selective enrichment in the spinal cord (GTEx). Its expression is greatly increased following oligodendrocyte differentiation (Cahoy et al., 2008).
PACS2 encodes an 889 amino acid multi-functional sorting protein that belongs to the PACS family of proteins. PACS2 mediates the trafficking of various cargo proteins between the ER, mitochondria, Golgi, lysosomes and plasma membrane, and has roles in both the cytoplasm and nucleus. In the cytoplasm, PACS2 regulates endoplasmic reticulum (ER) homeostasis, ER-mitochondria communication at mitochondria-associated membranes (Simmen et al., 2005; Youker et al., 2009) and the trafficking of ion channels to distinct subcellular compartments (Köttgen et al., 2005). PACS2 also binds the multi-functional 14-3-3 proteins to promote lipid biogenesis and inhibit apoptosis (Aslan et al. 2003). In the nucleus, PACS2 is involved in modulating the DNA damage response by regulating SIRT1 nuclear gene expression (Thomas et al., 2017).
The PACS2 protein contains three domains: a cargo (furin)-binding domain (FBR), a disordered middle-region (MR) and a C-terminal domain (CTR) whose function is unknown. The FBR domain binds cargo proteins for trafficking to various organelles. The MR contains a nuclear localization signal, an autoregulatory domain which includes a phosphorylatable acid cluster of amino acids that binds the FBR to access client proteins, and a binding site for the multi-functional 14-3-3 proteins.
The disease mechanism of PACS2-related syndrome is poorly understood but it is thought that the E209K variant, which is located in the autoregulatory domain of PACS2, results in a gain-of-function phenotype that alters the binding of PACS2 to one or more of its client proteins that are critical for processes related to neuron communication and cerebellar development (Olson et al. 2018). Co-immunoprecipitation experiments in human cell lines and patient-derived fibroblasts cell lines revealed that several PACS2 client proteins (histone deacetylases SIRT1, SIRT2, HDAC2 and ion-channel TRPV1) and 14-3-3εinteract with E209K mutant PACS2 more avidly than with the wild-type protein (Olson et al., 2018; Thomas & Villar-Pazos, 2020; Zang et al. 2022). The E209K PACS2 protein exhibits a slower turnover rate in human cells which may contribute to altered protein interactions (Zang et al. 2022). Taken together, these findings suggest that the PACS2 E209K variant is a gain-of-function mutation that may disrupt several biological processes that are critical for neurodevelopment through the dysregulation of client proteins. However, we anticipate that additional functions of PACS2 will be elucidated, e.g., the role of the disordered region, which will require revising the model of PACS2 E209K pathophysiology.
Another member of the PACS family, PACS1, is a paralog of PACS2 whose function also involves binding client proteins and directing them to various subcellular locations. The canonical 963-amino acid PACS1 shares 54% sequence identity with PACS2 and their FBRs share nearly 80% sequence identity. Mutations in the PACS1 gene give rise to PACS1 syndrome, a rare neurodevelopmental disorder that shares overlapping clinical features with PACS2 syndrome (Schuurs-Hoeijmakers et al., 2012). The majority of the nearly 100 reported individuals with PACS1 syndrome have the same de novo missense variant c.607C>T (p.R203W) which is proposed to result in either a gain-of-function or a dominant-negative mechanism (Arnedo et al., 2022). Ongoing research and therapy development by the PACS1 Syndrome Research Foundation offers a useful framework which can be applied to developing treatments for PACS2 syndrome and potential collaborators for research initiatives.
Given that PACS2 is a multi-functional protein involved in several diverse biological processes, and its disease mechanism is poorly understood, we envision advancing toward new therapeutic approaches by generating and characterizing cellular-based disease models that enable unbiased drug repurposing and targeted antisense oligonucleotide (ASO) therapeutic strategies.
To date, the PACS2 Research Foundation has generated patient-derived fibroblasts and iPSCs, engaged with key players in the field of PACS-related research to explore collaborative research opportunities, and Lena’s application has been accepted by the n-Lorem Foundation for personalized ASO therapy development. We will use the initial groundwork carried out by the foundation as a launchpad for the Cure Roadmap.
The development of disease models is necessary to gain insight into pathogenic mechanisms underlying PACS2 syndrome and identify potential therapies. To date, research into PACS2 mostly involves the clinical characterization of PACS2 syndrome or basic science to elucidate the protein’s function, necessitating the development of new models that can be employed across therapeutic tracks. However, some experiments have been carried out with patient-derived fibroblasts which have resulted in the identification of screenable morphological phenotypes and offered additional evidence for the proposed gain-of-function mechanism of the E209K variant. A Pacs2E209K mouse model has also been generated. We recommend the development and characterization of patient-derived fibroblasts and iPSC-neurons be pursued in parallel to support drug repurposing and antisense oligonucleotide therapeutic approaches. We also provide insight into the utility of model organisms such as flies, worms and zebrafish in these therapeutic approaches.
Patient-derived fibroblast lines
Patient-derived fibroblast lines are a valuable cellular model for elucidating disease mechanisms and can be readily employed for high-throughput drug (and genetic modifier) screens following the development of a suitable cell-based screening assay.
Limited studies have been carried out in PACS2 syndrome patient-derived fibroblasts. PACS2 E209K patient fibroblasts display a dispersed Golgi, a disorganized microtubule skeleton, and an increased interaction between the mutant protein and SIRT2. Small molecule inhibition or siRNA knockdown of SIRT2 was able to restore Golgi positioning in PACS2 patient fibroblasts (Thomas & Villar-Pazos, 2020). These preliminary data provide further evidence that the de novo E209K PACS2 variant is a gain-of-function mutation that results in increased interactions between PACS2 and several client proteins, and suggests that Golgi and microtubule phenotypes in PACS2 syndrome patient fibroblasts may be useful rescue readouts for drug screens. However, it is worth pointing out that these findings have only been reported in a patent and have not undergone peer review in a scientific publication.
Lena’s fibroblasts, as well as fibroblasts from her unaffected fraternal twin and both of her parents, have been generated at the Coriell Institute and are now publicly available to order. Lena’s fibroblast line is currently the only PACS2 syndrome cell line publicly available for research use. We recommend that additional PACS2 syndrome patient fibroblast lines be generated either through Coriell (~12-month timeline) which can be done at no cost to families, or via a private biobank.
Because PACS2 is a multi-functional protein that interacts with many client proteins, impinges upon numerous biological processes, and there is limited knowledge of the disease mechanisms underlying PACS2 syndrome, we propose that Lena’s fibroblasts be characterized by unbiased morphological profiling to discover image-based phenotypes which may be amenable to high-throughput drug screening. Cell Painting is a well-established high-content image-based assay that uses multiplexed fluorescent dyes to label different compartments of the cell such as the nucleus, endoplasmic reticulum, mitochondria, cytoskeleton, Golgi apparatus, and RNA. We recommend that Cell Painting of Lena’s fibroblasts be carried out at Charles River Laboratories which may confirm the previously reported Golgi and microtubule defects in PACS2 E209K patient fibroblasts, and potentially identify new morphological phenotypes. If phenotypes are identified, we recommend a continued partnership with Charles River Laboratories to develop a high-content screening assay that can be used in drug repurposing screens (outlined in Therapeutic Track 1).
Towards gaining insight into the disease mechanism of PACS2 syndrome and informing therapeutic strategies, it would be valuable to determine if the E209K mutant PACS2 forms protein aggregates with itself and/or other proteins by immunofluorescence. If multiplexing allows, it would also be valuable to determine the distribution and abundance of PACS2 client proteins at the same time as assessing the distribution and abundance of the PACS2 protein. These experiments could be carried out by an academic lab collaborating with PACS2 Research Foundation on other research initiatives. Note that the need may arise to develop a PACS2 E209K-specific antibody or to endogenously and allele-specifically fluorescently tag PACS2 protein in order to fully elucidate the behavior of wildtype and mutant PACS2 proteins in the same cell.
Disease modeling using iPSCs is a powerful approach for exploring pathogenic mechanisms in neurological disorders as they provide the opportunity to work directly with human neurons. iPSC-derived neurons can be used to investigate the effects of the E209K variant and further our understanding of PACS2 syndrome as a neurodevelopmental disease. Furthermore, iPSC-derived neurons can be used to screen antisense oligonucleotides that knock-down E209K mutant PACS2 transcripts or to validate drug repurposing candidates.
Lena’s induced pluripotent stem cells (iPSCs) have been generated by iXCell Technologies and several vials will likely be banked at the WiCell Research Institute to enable research on PACS2 syndrome. Ideally, additional iPSC lines and CRISPR-corrected isogenic controls should be generated from PACS2 patients (and a control line from an unaffected family member).
PACS2 Research Foundation has already engaged in discussions with Dr. Alicia Guemez-Gamboa to launch a research initiative focused on generating and characterizing PACS2 syndrome iPSC-neurons. Dr. Guemez-Gamboa is a key player in PACS1 syndrome research and developed several neural models which have provided critical insight into the disease mechanisms underlying the disease (Rylaarsdam & Guemez-Gamboa, bioRxiv). We recommend that the Foundation engage in a collaborative research initiative with Dr. Guemez-Gamboa to generate and phenotype E209K iPSC-derived neurons generated from both Lena’s iPSCs and via CRISPR in healthy control lines. Generating and phenotyping a series of PACS2 neuronal lines (E209K/+, -/-, -/+, +/+) will be essential to delineate the effects of the E209K variant.
In parallel, we recommend generating Lena’s iPSC neurons in partnership with Everlum Bio to enable the screening of ASOs for therapy development (outlined in Therapeutic Track 2). Candidate ASOs can be further validated for the rescue of cellular phenotypes identified in the research collaboration with Dr. Guemez-Gamboa.
Although 2D cellular models are a useful tool for studying neurological disorders, they are limited in their ability to recapitulate the complexity of the human brain. 3D brain organoids are capable of recapitulating the various features and functionalities of the human brain and are thus a powerful approach to investigating the pathogenesis of neurodevelopmental disorders. For example, forebrain organoid models of PACS1 syndrome revealed that the R203W pathogenic variant acts in a gain-of-function manner to produce disease phenotypes that overlap with convergent features of autism spectrum disorder (Rylaarsdam & Guemez-Gamboa, bioRxiv). Similarly, brain organoid models could be useful for investigating the pathogenic mechanisms underlying PACS2 syndrome and help inform therapy development by providing insight into potential therapeutic targets or pathways.
The budding yeast Saccharomyces cerevisiae is widely used as a model organism for investigating human biology owing to its ease of genetic manipulation and the high conservation of essential cellular processes between yeast and humans. Yeast can be engineered into humanized patient avatars expressing any patient mutation and used in high-throughput drug screens, genetic modifier screens and genetic interaction screens. There is no PACS2 ortholog in yeast, so it is not a suitable disease model for PACS2 syndrome drug discovery. Although not critical to the success of the PACS2 syndrome Cure Roadmap, a humanized yeast model which expresses the human PACS2 gene with the E209K variant, could be used to investigate conserved protein interactions and gain-of-function disease mechanisms.
Fly and worm
Fly and worm model organisms are valuable research models for drug repurposing screens which can typically be generated and phenotyped at low-cost on a quick timeline. Invertebrates possess a single PACS gene that is likely involved in membrane trafficking (Thomas et al. 2017). The C. elegans PACS protein has been shown to localize to early endosomes at the pre-synaptic terminus of the neuromuscular junction where it mediates synaptic transmission (Sieburth et al., 2005) However, it does not contain a nuclear localization signal so it cannot localize to the nucleus as it does in vertebrates. A C. elegans model of PACS1 syndrome (owing to the high conservation of the mutation-containing domain) has been generated and preliminary studies have revealed a synaptic transmission phenotype (Rylaarsdam et al., 2022). In the case of PACS2 syndrome, the glutamic acid (E) 209 amino acid is not conserved in D. melanogaster (KrT95D gene) and C. elegans (pacs-1 gene) PACS genes. For this reason, we advise that the generation of fly or worm models to model PACS2 syndrome be deprioritized at this time. It may be worth reaching out to the Center for Precision Animal Modeling at the University of Alabama Birmingham to see if they would be able to create worm and/or fly PACS2 avatars.
Zebrafish are a useful model system for studying the pathophysiology of neurodevelopmental disorders as the developing nervous system can be visualized in transparent embryos while their large progenies make them amenable to high-throughput drug screening. Zebrafish contain PACS2 and PACS1 orthologues making them a suitable model for studying PACS-related neurodevelopmental disorders. To date, no zebrafish model has been generated for PACS2 syndrome caused by the E209K mutation although knockout and overexpression models have been characterized (Fu et al., 2008). E209 is conserved in the zebrafish PACS2 gene, and thus a heterozygous E209K mutant zebrafish model could be generated by a CRO specializing in generating zebrafish models such as ZeClinics. Characterization of both a homozygous PACS2 knockout zebrafish and an E209K heterozygous mutant model would provide valuable insight into the effects of the E209K mutation, such as evidence to support (or reject) the proposed gain-of-function mechanism. If the E209K heterozygous mutant model displays phenotypes consistent with PACS2 syndrome patients, it could be used as a model for validating hits from cell-based drug repurposing screens.
However, the generation of a zebrafish model is not critical to the success of the PACS2 syndrome Cure Roadmap (particularly for drug repurposing) and will require additional dedicated funds to generate, phenotype and screen the model. Our recommendation is to deprioritize the generation of zebrafish models until the feasibility of developing a robust cell-based phenotypic assay for drug screening is determined.
Mouse models have provided insight into the function of PACS2 as well as its role in human metabolic and neurodegenerative diseases. For example, aberrantly elevated expression of PACS2 and enhanced MAM formation are found in Alzheimer’s mouse models and neurons from Alzheimer’s patients, suggesting that dysregulated expression of PACS2 plays a role in the disease pathology (Hedskog et al. 2013). Earlier this year, the first mouse model of PACS2 syndrome (Pacs2E209K/+) was generated by Dr. Gary Thomas’ lab, however, it is unknown if phenotyping studies are planned in the lab for the near future. The characterization of this model should enable a greater understanding of the pathophysiology of E209K variant and if disease relevant-phenotypes are observed, will be an essential resource for preclinical studies.
However, preclinical evaluation of antisense oligonucleotide therapies typically requires the generation of a humanized mouse model, ranging from the insertion of the entire human gene or a specific region. We recommend the generation of humanized Pacs2 E209K/+ mouse model at the Jackson Laboratory be pursued alongside ASO therapy development.
We propose two therapeutic tracks, drug repurposing and antisense oligonucleotide (ASO) therapy, to be pursued in a parallel and proteolysis-targeting chimeras (PROTACs) be explored as a third potential therapeutic track.
Therapeutic Track 1: Drug Repurposing
Drug repurposing is a strategy for identifying a new therapeutic use for an existing approved drug. This strategy has many advantages over developing a new drug for a given indication, particularly for rare and ultra-rare diseases. Repurposing candidates have already demonstrated safety in preclinical models and at minimum, early-stage clinical trials, and are thus less likely to fail in efficacy studies due to safety concerns. Additionally, the timeline and cost for drug development can be reduced as pre-clinical testing and safety assessment can be bypassed.
Following the development of a high-content cell-based assay for PACS2 syndrome (as outlined in the disease models section), we propose a fluorescent-image-based phenotypic drug repurposing screen be carried out using Lena’s fibroblasts to identify compounds that can restore the phenotype. Ideally, this assay would involve staining one or more subcellular compartments (such as Golgi and microtubules). We recommend the screen be carried out by Charles River Laboratory using the Broad Repurposing (REPO) Hub library. The REPO library has been recently expanded and consists of 6,808 compounds which have been either FDA or globally approved (2,502 compounds), tested in clinical trials (1,986 compounds) or evaluated in preclinical studies (2,313 compounds). Following the identification of top repurposing candidates, it would be beneficial to validate repurposed hits in additional PACS2 syndrome patient-derived fibroblast lines. Alternatively, repurposed candidates could be further validated in this disease-relevant cell model such as iPSC neurons.
Therapeutic Track 2: Antisense oligonucleotides (ASOs)
Antisense oligonucleotides (ASOs) have the potential to modulate gene expression by complementary base pairing to the mRNA of the targeted gene. ASOs can be used to promote exon inclusion or exclusion to restore the production of a functional or partially functional protein or to induce protein knockdown by targeting mRNAs for degradation. Because of their high-sequence specificity, ASOs can be designed as allele-specific or non-specific (i.e. allele-agnostic).
Based on the initially reported findings that suggest the de novo E209K mutation results in a gain of toxic function, an allele-specific ASO approach could be employed to improve the symptoms associated with PACS2 syndrome. This approach would allow the expression of the wild-type copy of the PACS2 gene to be maintained while knocking down the mutant copy. As the PACS2 gene is intolerant to LoF mutations (pLI=1; GnomAD) and homozygous PACS2 knockout mice display neurological and behavioral phenotypes, as well as limb defects (short tibia), we recommend deprioritizing an allele-agnostic approach.
The n-Lorem Foundation specializes in developing ASO medicines for individuals with “nano-rare” disease (< 30 patients) and provides the therapies to the patients for free, for life. Families must apply and undergo a review process to determine if they are a good fit based on several criteria. In April 2022, Proposal for Treatment to the n-Lorem Foundation on behalf of Lena and her family was submitted.
Alternatively, we recommend pursuing the development of an allele-specific ASO by an alternative path such as partnering with a biotech company like iXCells Biotechnologies which specializes in in vitro ASO screening and has partnered with a number of rare disease foundations. We propose up to 50 ASOs be designed and recommend partnering with Everlum Bio to design the top ASO candidates. One approach is to design ASOs that can be utilized in both in vitro and in vivo models (i.e. a humanized Pacs2 E209K/+ mouse). As described in the disease models section, the family is planning to partner with an academic lab to phenotype Lena’s iPSC-neurons. Restoration of these phenotypes can be used to validate ASO candidates by demonstrating function improvements. The traditional path for evaluating ASOs for potency efficacy and safety involves testing ASOs in a humanized mouse model of the disease.
Therapeutic Track 3: Proteolysis-targeting chimeras (PROTACs)
Proteolysis-targeting chimeras (PROTACs) are an emerging therapeutic modality that harnesses the ubiquitin–proteasome system (UPS) for targeted degradation of disease-causing proteins. PROTACs are heterobifunctional small molecules that bind both a protein of interest and an E3 ubiquitin ligase. Simultaneous binding of the target protein and the ligase by the PROTAC induces ubiquitylation of the protein and degradation by the UPS. PROTACs have many advantages over traditional small molecule inhibitors, such as their potential to modulate disease-causing proteins that have been previously difficult to target by inhibitors, as well as their ability to eliminate a protein’s functions entirely, rather than blocking a part of its function. (Zhao et al., 2022). PROTACs also have several advantages over ASOs, as they can be developed for oral route of administration and crossing of the blood-brain barrier, thus minimizing the burden on patients and the potential for infection. Since the first PROTAC degrader entered clinical trials in 2019, several biopharmaceutical companies have TPD molecules in clinic development (Békés et al., 2022).
PROTACs are an attractive therapeutic strategy for treating PACS2 syndrome as it has the potential to clear the mutant PACS2 protein and thereby eliminate its function. However, as limited knowledge on the effect of the de novo Glu209 PACS2 mutations (i.e. is it a gain-of-function mutation) and the disease mechanism, key questions must first be answered:
● Does the mutant PACS2 protein form aggregates?
● Is the reduction of global PACS2 protein levels (both mutant and wild-type proteins) sufficient for therapeutic benefit? or;
● Is the elimination of only the mutant PACS2 protein required, necessitating the need to design mutant-specific PROTACs?
Perlara can broker introductory meetings with key opinion leaders or biopharmaceutical companies such as Kymera in the TPD space to further explore this therapeutic avenue.
This Cure Roadmap is considered version 1 (v1) and it will require annual comprehensive reviews to incorporate progress reports, course corrections and unexpected opportunities arising from newly discovered modalities.
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