Early detection of liver cancer has the potential to substantially improve survival rates. Yet, diagnosis of hepatocellular carcinoma (HCC) is hampered by poor sensitivity of traditional imaging techniques - such as ultrasound, CT, and MRI - which often miss small or atypical tumors, particularly in cirrhotic livers with altered anatomy. As a result, more than a third of early-stage HCCs are missed. Earli is addressing these limitations by developing a platform technology that utilizes LNP-delivered recombinant DNA constructs containing cancer-activated synthetic promoters driving the selective expression of proteins such as well defined epitopes for the imaging of tumors or cytokines that have therapeutic potential to treat the tumor. Although non-viral systems like lipid nanoparticles (LNPs) have proven effective for RNA delivery, DNA delivery to tumor cells remains challenging. Earli-derived LNP formulation FRM237 was used to encapsulate a recombinant DNA comprised of a non-specific CAG promoter to drive the expression of a firefly luciferase reporter and was evaluated by ex vivo bioluminescence imaging (BLI) in a subcutaneous Hep3B (liver adenocarcinoma) xenograft model. The FRM237 formulation produced high levels of expression and achieved BLI levels that were 5-fold higher than the same DNA formulated in a multi-component LNP used in a commercial siRNA product. More importantly, engineered LNP enabled 88-fold greater tumor-to-liver BLI ratio, revealing liver tumor tropism. Pharmacokinetic studies show a large increase in circulation LNP-delivered DNA vector copies were consistently 10-fold higher in serum as compared to the competitor formulation. Effective extrahepatic delivery and expression to HCC was further verified in an orthotopic liver xenograft model using a recombinant DNA driven by a synthetic promoter known to be highly active in liver cancer cells. Administered via IV injection to mice with Hep3B tumors, this product produced 96-fold higher expression from those malignant tissues as compared to naïve liver tissues. Likewise, immunohistochemistry (IHC) analyses revealed selective expression of the protein throughout the orthotopic Hep3B tumors with little or no corresponding staining in normal hepatocytes. Finally, intravenously dosed lead formulations were tolerated as evident by modest but transient body weight loss and clinical observations. Overall, our data collectively points to successful delivery of DNA to hepatic tumors via engineered LNP formulations following iv administration. When combined with cancer-activated expression of PET reporter genes and further validation in preclinical mouse models and toxicology studies, this represents a promising diagnostic imaging platform for early detection of liver cancer. Nadege Morisot, Blaine McCarthy, Sushil Lathwal, Dariusz Wodziak, Suthara Ramachandran, Anderson Lee, Dang Dang, Blair Cain, Xiaobin Wu, Robby Chandra, Badriprasad Ananthanarayanan, David Suhy, Christine Peterson. Nanoparticle delivery of cancer-activated DNA constructs for the diagnosis of liver tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 60.

Abstract Turning early-cancer diagnosis into actionable treatment often relies on highly accurate and sensitive imaging techniques to guide surgical intervention or to monitor therapeutic efficacy. Earli is developing a highly sensitive, orthogonal approach that uses genetic constructs to usurp dysregulated cancer pathways to force the tumor to produce a PET reporter gene, enabling the use of radiotracers amenable to positron emission tomography/computed tomography (PET/CT) to guide precision imaging. However, analysis of PET/CT images using standard manual and semi-automated methods is challenging resulting in reduced experimental throughput and user variability, highlighting the need for a fully automated PET/CT analysis pipeline using Deep Learning (DL). To increase pre-clinical throughput and reduce inter-user variability, we developed an automated DL processing pipeline in Python to perform three major tasks: separation of multi-mouse bed data for PET uptake quantification by mouse, segmentation of tumors and background organs, and quantification of PET signal for PK/PD analysis. Preclinical PET/CT data was acquired using a 4 animal, multi-mouse bed and separated into individual mice by co-registering the reconstructed CT image against a multi-mouse bed reference mask using a rigid 3D Euler transformation algorithm and cropping at fixed indices. Following mouse separation, a 3D nnUNet architecture is used to perform semantic segmentation of regions of interest (ROI) on CT for bi-hemispheric subcutaneous tumors, lungs, liver, kidneys, spleen, and the bladder. nnUNet was trained on PET/CT of 311 mice consisting of a mixture of naïve and tumor-bearing animals subcutaneously implanted with H1299 cells using a five-fold cross-validation strategy with 1000 epochs per fold. Model performance was evaluated by comparing the network prediction to the reference manually annotated masks using the Dice coefficient. PET uptake is reported as percent injected dose per milliliter (%ID/mL) or standardized uptake value (SUV). Segmentation performance results for the hold-out set identified mean Dice scores greater than 0.80 for all ROIs, reflecting the similarity between model predictions and human annotations. The automated pipeline reduced the analysis time from 5.5-6 hours per mouse when using traditional manual/semi-automatic approaches to approximately 15 minutes. In summary, we developed a fully automated DL-based PET/CT quantification pipeline for pre-clinical mouse studies that provides accurate ROI segmentation and PET uptake quantification, significantly increasing experimental throughput and reducing inter-user variability. Further improvements to model inference include loss functions weighted for poor performing ROIs and addition of lung nodule segmentation for eventual translation to humans as an accompaniment to Earli’s theranostic programs. Citation Format: Hung-Yu Henry Lee, Mohammed Goryawala, Tim Sproul, Maggie Louie, David Suhy. Leveraging deep learning for fully automated analysis of pre-clinical mouse positron emission tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3529.

Abstract Earli is developing a highly sensitive, orthogonal approach that is designed to be delivered via IV injection and uses a genetic construct to usurp dysregulated pathways, and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. The key element of our cancer-activated construct that drives specificity and sensitivity of expression is a promoter sequence, synthetically engineered to leverage transcription factors (TFs) activated in cancer pathways. Acting as a molecular sensor for dysregulated TF levels, the DNA construct expresses a measurable biomarker in cancer cells, while remaining transcriptionally silent in normal adjacent tissues and benign lesions. Previously, we described a multiomics factor analysis (MOFA) to interrogate 200 non-small cell lung cancer (NSCLC) and matched normal adjacent tissue samples comprised of RNA-seq based transcriptional profiling, and MS-based proteomics and phospho-proteomics from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset, and generated a list of top TFs dysregulated in NSCLC. In this study, we show the capability to design, engineer, and test cancer specific activity of combinations of response elements using a Massively Parallel Reporter Assay (MPRA) high-throughput pooled screening (HTS) method. More than 2,000 unique sequences of homotypic or heterotypic TF binding sites corresponding to the top 100 TFs were arrayed with diverse spacing and individually barcoded for the high-throughput screens in NSCLC cell lines. To validate our top hits, we individually tested 10 sequences from the top 25% and 8 sequences from the bottom 25%. Nine of the top sequences produced signals 5-10X higher than the background, whereas only one of the eight bottom sequences produced signal higher than the background. We have individually validated 30 different response elements and found that 28 of 30 drove the expected expression, with the majority of response elements (n=20) showing expression 5-10X higher than background. Remarkably, two of the engineered response elements were able to drive expression 20-30X higher in a specific cell line activated by WNT-signaling, suggesting that these response elements can sense specific cancer dysregulated pathways. Our lead response elements are being tested across a broad range of in vitro primary tumor lines with diverse genetic backgrounds and transcriptional profiles prior to in vivo testing using our imaging platform to distinguish malignant lung nodules. In summary, we have established a robust screening platform to identify response elements that can be activated by cancer dysregulated pathways. We are currently expanding this platform to other cancer models and indications, and leveraging this to explore more complex response elements that use combinations of TFs and natural spacing. Citation Format: Elizabeth Stroebele, Yue (Wendy) Zhang, Ishan Podar, Chloe Xia, Ajda Rojc, Dariusz Wodziak, Maggie Louie, David Suhy. Using high-throughput screening to identify DNA response elements that sense cancer dysregulated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1694.

Abstract Current modalities, such as LDCT and fluorodeoxyglucose positron emission tomography (FDG PET), for early lung cancer imaging, rely upon proxies resulting in poor specificity and/or sensitivity. They are also unreliable for discriminating benign versus malignant lesions resulting in the requirement of longitudinal analyses euphemistically called “watchful waiting”. Lung biopsies are marred by significant false negative rates and pose complications of collapsed lungs or bleeding in up to 22% of procedures and death rates up to 1%. To overcome these obstacles, Earli is developing a non-invasive, highly specific cancer-activated imaging platform that usurps dysregulated gene expression within malignancies to force only cancer cells to produce a synthetic biomarker that can be localized using PET imaging. EARLI-204 is a lipid nanoparticle that contains a DNA nanoplasmid comprised of a cancer-activated promoter driving selective expression of a functionally inactivated somatostatin receptor 2 (SSTR2fi) gene. Abrogation of internalization and signal transduction allows the safe use of SSTR2fi as an ectopically expressed PET reporter gene (PRG). Following administration, EARLI-204 transfects tumor and normal cells but transcriptional activation only within cancer cells results in selective cell-surface expression of SSTR2fi that can be imaged with FDA-approved SSTR2 PET tracers. H1299 human lung cancer cells engineered to express SSTR2 (H1299-SSTR2) revealed a discernable PET signal with as little as 31,000 engineered cells. Mixed cell titration studies using subcutaneous (SQ) tumors with H1299-SSTR2 and non-expressing H1299-wt cells suggest tumors with between 0.1% and 1% H1299-SSTR2 cells had a significant PET signal, compared to tumors with 100% H1299-wt. This result sets a benchmark transfection of ~1% to achieve a PET signal. Intratumoral (IT) dose titration of EARLI-204 revealed that 5 ug of EARLI-204 generates a significant PET signal. Consistent with the H1299-SSTR2 studies, IHC shows ~1% of tumor cells express the PRG at a 5 ug IT dose level. Furthermore, we measured 70 copies of EARLI-204 DNA per cell resulting in 22,629 SSTR2 mRNA copies/25 ng at a 5 ug IT dose level. IV dosing of EARLI-204 in SQ and intralung tumor models results in cancer-activated expression of SSTR2fi in both SQ and small (<4 mm) lung tumors with significantly increased (p < 0.05) PET signal. For lung tumors, analyses showed 1 copy of EARLI-204 DNA/cell resulting in 780 SSTR2 mRNA copies/25 ng. The results demonstrate an effective molecular imaging platform for drug discovery ranging from in vivo screening to building PK-PD-efficacy relationships and enabling human dose projection for the Earli PET localization drug. The benchmarks and findings established with this preclinical imaging pipeline are currently being evaluated in large animals such as dogs and pigs, and will be translated to humans in planned clinical trials. Citation Format: Mohammed Goryawala, Hung-Yu Henry Lee, Ling Tong, Trupti Patil, Alex Harwig, Chloe Xia, Suthara Ramachandran, Dariusz Wodziak, Dean Felsher, Tim Sproul, David Suhy. Development of a cancer-activated biologic imaging platform for early lung cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4151.

Early detection of cancer is an important driver of increased survival, quality of life and reduced healthcare costs. Earli is developing a highly sensitive, orthogonal approach that uses a genetic construct that usurps dysregulated pathways and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. Identification of abnormally elevated transcription factors (TF) in cancer is crucial when developing a cancer-activated expression platform. Evaluating TF function in a genome wide fashion is a challenge, especially since TF activity is not solely reflected by just its RNA expression or even by protein abundance, but also its post-translational modification including phosphorylation. In this study, we use the publicly available multiomics dataset from Clinical Proteomic Tumor Analysis Consortium (CPTAC) which includes RNA-seq based transcriptional profiling, MS based protein abundance and phosphorylation from 211 paired tumor and normal adjacent samples derived from NSCLC patients. Performing Multiomics Factor Analysis (MOFA) analysis, we identified a short list of 34 dysregulated TFs in NSCLC that displayed high expression levels in at least two of the three omics platforms relative to normal tissues, six of which scored highly across all three. A subset of candidate TF binding sequences were subcloned as multimers into expression constructs with a basal promoter and empirically evaluated in multiple cancer cell lines, including patient-derived xenograft (PDX) cell lines and normal cell lines.Transfection experiments demonstrated that these novel chimeric synthetic promoters could produce robust levels of expression in PDX-derived cell lines that was 10-20x higher than expression mediated by known cancer-activated promoters such as the endogenous survivin (BIRC5) promoter. Furthermore, levels achieved were only 3-4 fold lower than a control EF-1a promoter, one of the strongest promoters, typically used to drive constitutive expression in mammalian cells. Currently, a small subset of these chimeric constructs is being tested in murine tumor models and in large animal cancer models such as oncopigs. These experiments will further drive the development of this approach for use in early cancer detection. Citation Format: Yue Wendy Zhang, Shireen Rudina, Dariusz Wodziak, Chloe Xia, Maggie Louie, Albert Park, David Suhy. Using multiomics analysis to identify dysregulated transcription factors in non-small cell lung cancer (NSCLC) to drive the expression of a cancer-activated synthetic biomarker. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4311.

Early detection and localization of primary cancers has been shown to improve clinical outcomes and overall survival. While liquid biopsy may indicate the presence of cancer and possibly provide the tissue of origin, it cannot pinpoint the exact location of cancer and its metastases. Furthermore, current imaging techniques lack the sensitivity to locate early-stage tumors, diminishing the ability for early clinical intervention. Current imaging approaches using 18F-FDG rely on increased metabolic rates of tumors vs. surrounding non-tumor tissue, often resulting in high background and inability to detect low-metabolic tumors. Earli is developing a highly sensitive, orthogonal approach that uses genetic constructs to usurp dysregulated cancer pathways to force the tumor to produce a synthetic biomarker used to detect and localize the malignancy. We previously demonstrated the utility of cancer-activated expression of blood-based biomarkers in preclinical cancer models, which are currently in an ongoing clinical study. EARLI-201 is novel genetic imaging probe comprised of a DNA construct containing a cancer-activated promoter to drive the expression of a PET Reporter Gene (PRG). The HSV-sr39TK PRG product specifically sequesters the 18F-FHBG tracer inside the cell, enabling detection by PET imaging. Initial studies for sensitivity combined H1299 lung cancer cells engineered with only two copies of the PRG were implanted subcutaneously into immunocompromised mice followed by the administration of the 18F-FHBG tracer. Results demonstrate a robust signal from as few as 62,000 injected cells. Given that a 1 mm3 tumor contains ~1,000,000 cells, suggests that we can localize tumors as small as 6.25mm3 with only a 1% transfection efficiency. EARLI-201 was tested in a variety of murine models including the metastatic “Lung Trap” and spontaneous HCC-MYC/Twist1, which produce discrete tumor nodules in the lungs or liver, respectively. A single intravenous injection of EARLI-201 given at Day 1 results in cancer-activated expression and accumulation of the PRG in tumors. 18F-FHBG PET imaging three days post dosing revealed distinct tumor foci that co-register with tumors detected by CT. Additional ex vivo techniques confirmed PRG expression only in tumor-containing tissues and not in healthy tissue. Imaging the same animals with 18F-FDG failed to produce any appreciable signal in the same tumors, likely because they are of low metabolic nature. These pre-clinical results clearly demonstrate the potential of Earli’s approach to detect and localize very small tumors currently outside the sensitivity of existing imaging modalities. Additional experiments to evaluate EARLI-201 in companion dogs diagnosed with cancer are currently in progress and provides the ability to validate the platform in larger body masses with spontaneously formed tumors. Citation Format: Alex Harwig, Morgan Wang, Ling Tong, Regina Nieu, Vy Nguyen, Dean Felsher, David Suhy. A more sensitive approach to cancer imaging using cancer-activated PET reporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2480.

Early detection of cancer is an important driver of increased survival, quality of life and reduced healthcare costs. Although liquid biopsy provides a new option for early cancer detection, the limitations of detecting a few molecules of blood-based biomarkers naturally shed by the cancer remain. Earli is developing a highly sensitive, orthogonal approach that uses a genetic construct that usurps dysregulated pathways and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. We previously used murine xenograft models to validate the tumor detection properties of EARLI-001, a circular DNA containing a human derived cancer-activated promoter to drive the transient expression of secreted embryonic alkaline phosphatase (SEAP), a protein normally expressed only during fetal development. Biodistribution following intravenous (IV) administration of EARLI-001 shows a broad tissue tropism, enabling the potential to monitor multiple tissues for malignant cells. A single dose of EARLI-001 administered in a pulmonary metastatic cancer mouse model resulted in a 95-fold increase in serum SEAP compared to healthy controls. Recognizing the inherent limitations of murine models, we evaluated EARLI-001 in canines diagnosed with cancer. Dog cancers share many features with human malignancies including spontaneous tumor formation, heterogeneity, growth kinetics, histology, and comparable dysregulated genetic pathways. Furthermore, the use of dogs allows the study of EARLI-001 in an immune competent setting and with substantially increased size (up to 65 kg) comparable to scaling into human subjects. EARLI-001 was first tested for safety and biodistribution in 54 purpose-bred beagles in MTD and GLP toxicology studies which reveal that it is safe and well tolerated across a broad range of clinical doses. Biodistribution analysis in 12 tissues showed nanoplasmid levels peaking in the first days post dose and rapidly decreased to background levels by 60 days. These non-tumor bearing dogs showed no serum SEAP signal at all doses. Following IACUC approval and informed owner consent, EARLI-001 was administered to companion dogs in a standard 3 x 3 dose escalation study, with an expanded mid-range cohort. These companion dogs harbored a wide variety of spontaneous tumors in different tissues of origin. In tumor bearing dogs, EARLI-001 elicited a clear and discernable cancer-activated SEAP signal in 1 of 3 dogs dosed at 0.01 mg/kg, 3 of 6 dosed at 0.03 mg/kg, 2 of 2 at 0.07 mg/kg and 8 of 8 dogs dosed at 0.1 mg/kg. Furthermore, the MTD was not reached, and no significant dose-limiting toxicity was observed in these older, disease-burdened animals. These results show the utility of the platform to interrogate tumor biology and detect the presence of malignancies in relevant in vivo models and provided the critical safety and efficacy data towards a first-in-human clinical study in lung cancer initiated in 2021. Citation Format: Regina Nieu, Emily Phenix, Jennifer Hauss, Hadley Hanson, Nga Ho, Alex Harwig, Shireen Rudina, Badri Ananthanarayanan, Hong Chang, Bijee George, Maggie C. Louie, Julie Bulman-Fleming, Michael Kent, David Suhy. Detection of cancer in dogs using a novel genetic-based synthetic biomarker platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3382.

The expression of synthetic biomarkers from genetic constructs represents a new paradigm of molecular diagnostic tools for early cancer detection and localization. One of the limitations of liquid biopsy approaches is the low abundance of endogenous biomarkers shed in early-stage disease. To overcome this, Earli’s platform instead uses genetic constructs to force tumors to produce cancer-activated synthetic biomarkers. Thus, technologies that deliver DNA to tumor cells in multiple tissues need to be developed. While non-viral delivery systems such as lipid nanoparticles (LNPs) have achieved clinical success for RNA, the delivery of DNA to tumor cells remains a challenge. We have engineered two classes of nanoparticles using biodegradable and ionizable cationic materials. The efficacy of these nanoparticles to deliver a DNA nanoplasmid containing a cancer-activated promoter to drive expression of a secreted embryonic alkaline phosphatase (SEAP) was evaluated in multiple cell-derived xenograft (CDX) models. The first class of nanoparticles was comprised of poly-beta-amino-ester polymers (PBAEs). Systematic screening of a library of PBAEs and optimization of PEG-lipid content produced nanoparticles approximately 80-100 nm in diameter with slightly positive zeta potential, enabling biodistribution to mouse lung tissues. Testing of initial nanoparticle compositions identified safe and effective formulations that produced 5-fold higher SEAP expression levels in lung tumor-bearing animals versus naïve mice. Further enhancements produced SEAP levels up to 250-fold higher in tumor-bearing versus control mice but resulted in significant increases in liver enzymes, suggesting tolerability issues that are currently being addressed by improving the polymer and formulation composition. The second class of nanoparticles was composed of ionizable lipids such as DLin-MC3-DMA that have been clinically validated for RNA delivery to the liver. Optimized compositions with helper lipid and PEG-lipid variants identified formulations that could successfully deliver DNA to the liver. These formulations produced 30-fold higher SEAP expression in tumor-bearing animals compared to naïve controls in an H1299 CDX model. A 7-fold difference in SEAP levels was demonstrated in an orthotopic Hep3B CDX model between tumor versus non-tumor bearing animals. Strikingly, LNP delivery of a cancer-activated Renilla luciferase construct produced tumor-specific bioluminescence, demonstrating the ability of the LNPs to localize liver tumors. LNP formulations were well tolerated, producing transient, mild to moderate elevations in liver enzymes one day after administration that subsequently resolved. These potent and safe nanoparticle delivery systems are promising agents for enabling future clinical translation of our synthetic biomarker platform. Citation Format: Badri Ananthanarayanan, Regina Nieu, Weihang Ji, Blaine McCarthy, Shengshuang Zhu, Alex Harwig, Maggie C. Louie, David Suhy. Nanoparticle delivery of cancer-activated DNA enables the detection and localization of tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1732.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codonoptimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

Abstract Early detection of cancer is a powerful driver of increased survival rate, quality of life and reduced healthcare costs. ‘Liquid biopsies' that rely upon chemistry for the detection of blood-based endogenous cancer biomarkers such as proteins, circulating tumor DNA or RNA show only limited sensitivity for certain early stage cancers where the tumor mass shedding those biomarkers is smaller. Rather than searching for elusive natural biomarkers shed into the bloodstream, we are employing a promising alternate approach rooted in biology to usurp the highly dysregulated transcriptional pathways that give rise to malignancies to force cancer cells to produce easily detectable ‘synthetic' biomarkers that do not naturally occur. EARLI-001 is a DNA nanoplasmid comprised of a cancer-activated promoter to drive expression of Secreted Embryonic Alkaline Phosphatase (SEAP). The survivin promoter was initially selected for proof of concept because the corresponding anti-apoptotic gene product is widely overexpressed in many cancers, including melanoma, liver, breast, lung, colon and ovarian cancer, while not being expressed in normal adult tissues. The synthetic biomarker SEAP is a secreted variant of human placental alkaline phosphatase, which is only expressed during fetal development and thus has near-zero background in adult blood. EARLI-001 is formulated using a non-viral linear polyethyleneimine and administered intravenously to enable broad tissue distribution and transient transfection of multiple tissues. The sensitivity and specificity of EARLI-001 for tumor detection were established using a variety of cell-derived xenograft models. A single administration of EARLI-001 in immunocompromised mice bearing a modest burden of lung metastases drove cancer-activated SEAP expression that was at least 100-fold higher than the corresponding expression in non-tumor bearing animals. In an immune-competent syngeneic model, EARLI-001 produced 10-fold higher SEAP expression in mice bearing lung metastases of 4T1 mammary tumor cells than naïve mice. EARLI-001 could also discriminate mice bearing orthotopic Hep3B liver xenograft tumors from naïve control mice. Finally, the sensitivity of EARLI-001 in detecting small tumors was demonstrated in a longitudinal study, in which robust cancer-specific SEAP expression occurred even when the lung tumor burden was too low to result in significant changes in organ weight. The toxicology, pharmacokinetics, and biodistribution of EARLI-001 were evaluated in several GLP studies in mice as well as canines, enabling translation to a first-in-man clinical study. This Phase 1 dose escalation study will evaluate the safety, tolerability and PK/PD of EARLI-001 in subjects with locally advanced or metastatic lung cancer and enable further clinical development of the cancer-activated synthetic biomarker platform. Citation Format: Badriprasad Ananthanarayanan, Regina Nieu, Evan Bishop, Shireen Rudina, Alex Harwig, Bijee George, David Suhy. Preclinical development of EARLI-001, a genetic platform producing cancer-activated synthetic biomarkers for the early detection of malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2548.

1 Centres of Gene and Cell Therapy and Biomedical Sciences, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, UK 2 Sorbonne Université, INSERM, Association Institut de Myologie, Centre de Recherche en Myologie, UMRS974, 47 bd de l’Hôpital, 75013 Paris, France 3 Comparative Biomedical Sciences, Royal Veterinary College, London, UK 4 Benitec Biopharma, Hayward, California, USA

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV 'silence' and 'replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the 'silence' and 'replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.