The American Society of Gene and Cell Therapy’s (ASGCT) Annual Meeting is the premier event for researchers in gene and cell therapy. The meeting will cover a range of topics from cutting-edge studies in cell and gene therapy, viral hepatitis, liver toxicity, MASH/MAFLD, and more.
PhoenixBio is excited to be sponsoring a booth and attending ASGCT's 27th Annual Meeting in person at the Baltimore Convention Center in Baltimore, MD on May 7th - 11th, 2024. Meet with our team at Booth #1917 to learn more about PhoenixBio's capabilities and how we can help you advance and deliver tomorrow’s therapies.
While you're here, be sure to attend humanized liver chimeric mouse model related abstracts featured below and grab a chance to speak with our team!
Humanized Liver Related Abstracts:
- #487. Title: A Comparative Analysis of Engineered Liver Tropic Capsids Across Different Species: Towards Cross-Species Translation. Abstract details: Rapid advancements in genomic medicine, particularly in viral vector-based delivery systems, underscore the necessity for AAV capsids suitable for translation from rodent models to humans. Therefore, there is a high need for a robust platform to assess the tropism and potency of engineered capsids. In our study, we focused on liver-tropic capsids and conducted a comprehensive analysis across diverse models, including ex-vivo primary human hepatocytes, in vivo wild-type (WT) mice, different liver humanized mouse models (FRG mouse versus PXB-mouse), and non-human primates (NHP), to evaluate the transduction efficiency of different liver-specific capsids designed for genomic medicine applications. In primary human hepatocytes, WT mice, and humanized mouse models, we employed reporter genes GFP and human FIX to evaluate transduction efficiency in different models. Droplet digital PCR measured GFP RNA expression levels, GFP protein was quantified by IHC staining, and ELISA was used to measure human FIX protein levels. In the NHP study, we barcoded different capsids and validated transgene RNA levels quantified by next-generation sequencing (NGS). Consistent results were observed for capsid ranking in two different liver humanized mouse models, FRG (FAH KO) and PXB-mice (cDNA uPA/SCID), aligning with the outcomes in human primary hepatocytes. The FRG mouse had roughly 50% human hepatocyte engraftment, while the PXB-mouse had 90% human hepatocyte engraftment. Notably, the top capsid from human models also demonstrated excellent transduction efficiency in the WT mouse liver, suggesting its potential use in preclinical, primate, and human studies. Importantly, the lead capsid identified in the WT mouse study, humanized mice, and primary human hepatocytes remained the top performer in the NHP study, the most relevant model to humans for liver-tropic capsids. The results of this comparative analysis not only deepen our understanding of capsid behavior but also lay the groundwork for a standardized screening platform for capsids, streamlining preclinical testing across diverse species. The identification of a common capsid with tropism and transduction capabilities across various species represents a significant advance in the field of genomic medicine.
- #15. Title: Reduction in Triglycerides through a Novel Ultracompact CRISPR System: Efficacy in Mouse Models and NHP Studies. Abstract details: In the last decade, CRISPR-based therapies have made tremendous progress in cell therapy applications and selected diseases that can be targeted in the liver. However, correcting the vast majority of genetic diseases outside the liver has remained challenging due to the large size of the original CRISPR systems, such as Cas9 and Cas12a. Ultracompact CRISPR systems present an opportunity to overcome the challenge of in vivo delivery, enabling single-AAV delivery of genome editors, including next-gen editing techniques such as epigenetic editing, reverse transcriptase editing, and base editing. Although many compact CRISPR systems have been described, their application to therapeutic targets has been limited due to challenges in matching the efficacy of larger systems. Here, we describe the discovery of a novel ultracompact CRISPR platform and show it is a potent genome editor in vivo in mouse and nonhuman primate models. Through high-throughput screening of natural variants, extensive protein engineering, and guide optimization, we optimize this platform for robust editing with both AAV and LNP delivery. This ultracompact CRISPR system was effectively used to target the APOC3 gene, a key player in triglyceride metabolism, offering promising therapeutic potential for durable treatment of severe hypertriglyceridemia. In vivo studies in humanized mouse models demonstrated an 80% reduction in triglyceride levels. Complementing these findings, our nonhuman primate studies further confirmed significant genetic editing of APOC3 with no major safety concerns observed. In addition, we provide an update on leveraging these ultracompact CRISPR systems for single AAV delivery of epigenetic editing for muscular dystrophy. These results underscore the potential of ultracompact CRISPR systems for precise and efficient gene editing applications in diverse conditions and to enable single-aav compatible next-gen editing approaches such as epigenetic editing.
- #56. Title: Compact Epigenetic Modulators for CRISPR Mediated Persistent Gene Activation. Abstract details: CRISPR-mediated epigenetic gene silencing is a powerful tool with significant therapeutic potential, due to its ability to induce long-lasting changes in gene expression without introducing mutations to genomic DNA. This holds great promise in the field of genetic medicine, potentially allowing for the treatment of complex human diseases. However, the lack of tools for robust and persistent activation of target gene expression remains a significant obstacle to epigenetic editing. To overcome this limitation, at Epic Bio we have established a screening platform to discover novel epigenetic activators. Through this platform, we have discovered multiple Gene Expression Modulation System (GEMS) activators capable of inducing persistent gene activation following transient delivery. In initial studies, GEMS activators containing a vIRF2 core domain (vCD) outperformed dCas-VPR (a fusion of VP16, P65 and Rta) and other potent activators in terms of gene activation potency, robustness, and persistence after transient delivery, in both immortalized and primary cells. This was observed when GEMS were transiently delivered to target genes in multiple cell types and chromatin states (from quiescent to highly expressed). Notably, novel hypercompact activators (64 to 98 amino acids) derived from vCDs demonstrated durable activation of IL-21, an interleukin crucial for enhancing CAR-T cell function, lasting for 41 days after transient expression in Jurkat cells. These GEMS activators achieved superior potency when compared to a benchmark transcriptional activator VPR. Initial potencies of vCD and VPR were ~730-fold and ~4 fold respectively, indicating that vCD outperforms VPR by a factor of ~177-fold. Throughout this period of gene activation, the vCD maintained an IL-21 activation plateau of ~30-fold, whereas VPR, in contrast, was unable to sustain IL-21 activation beyond 11 days. In hepatocyte HepG2 cells, flow cytometry studies were performed to assess the ability of vCDs to activate LDLR (Fig.1 a), a gene of therapeutic significance as LDLR haploinsufficiency constitutes 85 to 90% of genetically confirmed cases of familial hypercholesterolemia. We compared the activation potential of vCDs against VP64 and found that vCDs, but not VP64, were able to activate LDLR to levels approaching those achieved by transfection of an LDLR-encoding ORF cDNA (Fig.1 b). This activation was sustained in HepG2 cells for at least 16 days post-transient plasmid delivery. We then performed a direct comparison between vCDs and the potent activator VPR and demonstrated that vCDs were able to induce LDLR activation that persisted for 70 days post transient delivery in HepG2 cells (Fig.1 c).In vivo testing of vCD activators further demonstrated their efficacy and persistence. Modulating the expression of LDLR in primary human hepatocytes within a humanized mouse liver model, we achieved two-fold activation of LDLR, sustained for up to 5 weeks post-single dose administration of vCD GEMS activators encapsulated in lipid nanoparticles (LNPs), far beyond any previous report of in vivo CRISPR-mediated epigenetic gene activation (Fig.1 d-g). These results validate proof of concept for CRISPR-mediated persistent gene activation in vitro and in vivo, showcasing the potential of epigenetic activation of disease-modifying genes in vivo.
- #58. Title: Durable and Specific Silencing of Therapeutically Relevant Genes Using Epigenetic Editors is Reversible In Vivo. Abstract details: Epigenetic editing is a powerful approach designed to durably silence or activate genes by leveraging nature’s endogenous cellular mechanism for gene regulation without the inherent genotoxic risks of other editing approaches that nick or cut the DNA. Our epigenetic editors have a distinct mechanistic advantage by utilizing DNA methylation and demethylation to silence or activate genes, respectively, a targeted approach for regulation of gene expression rather than indirect regulation through cutting or nicking the DNA, which can cause unintended chromosomal effects. We have identified epigenetic editors (EEs) that have demonstrated efficient silencing activity against human hepatitis B virus (HBV) and PCSK9 in primary human hepatocytes (PHH) and mice. To characterize the specificity of epigenetic editing, we profiled PHHs treated with either HBV-EE or PCSK9-EE by RNAseq, methylation array, and whole-genome methylation sequencing. No statistically significant differentially expressed off-target genes or methylated regions were identified, confirming the specificity of our EEs in PHHs. We then evaluated the durability of epigenetic editing in transgenic mice expressing HBV and showed potent reduction (>2-log) in circulating HBsAg with durability out to 5 months. Similarly, transgenic mice carrying the human PCSK9 genomic locus treated with PCSK9-EE showed >98% reduction in circulating PCSK9 over 300 days. In addition, we confirmed that the efficient PCSK9 silencing observed after treatment with PCSK9-EE was fully maintained following a 2/3 partial hepatectomy, an established model of liver regeneration. Furthermore, targeted CpG methylation at the PCSK9 promoter region was highly correlated with PCSK9 expression and faithfully maintained in the regenerated liver of partially hepatectomized mice. Finally, we explored the reversibility of epigenetic editing in vivo by using an epigenetic activator that was identified for its ability to restore PCSK9 expression in previously silenced cells in vitro via targeted CpG demethylation. Mice that had been previously silenced with PCSK9-EE were treated with a PCSK9 epigenetic activator, resulting in a near-complete restoration of PCSK9 levels to baseline levels within 14 days after treatment and maintained for at least 56 days. Taken together, we believe our data further support the utility of epigenetic editors to specifically, durably and reversibly silence therapeutically-relevant target genes in the liver without altering the DNA sequence