CMN Weekly (22 November 2024) - Your Weekly CRISPR Medicine News

Top picks

• The First CRISPR Treatment Is Making Its Way to Patients. Nearly a year after its approval, CASGEVY, which became the first approved CRISPR therapy, developed to treat sickle cell disease and beta thalassemia, is finally reaching patients. Read more in this writeup for WIRED.
• CRISPRMED25 is an event not to be missed, but don't take it from us! Have a listen to the testimony of Colette Rogers, Ph.D., who attended last year's conference and received one of the Best Poster Awards! Check it out here.

Research

• In an article published yesterday in Molecular Therapy, researchers in Denmark report the remarkable versatility of CRISPR/Cas-based systems for genetic engineering. By combining transcriptional activation (CRISPRa), interference (CRISPRi), and gene knockout, the team, led by Rasmus Bak at Aarhus University, established a streamlined approach for trimodal genetic engineering. They propose that their one-step, non-viral engineering approach provides a foundation for more sophisticated applications in regenerative medicine and therapeutics, offering transient transcriptome modulation alongside permanent genetic changes.
• A team of researchers at New York Genome Center and New York University have explored how the loss of more than 6,000 human long non-coding RNAs (lncRNAs) impacted cellular fitness in immortalised human embryonic kidney cells and four cancer cell lines. The massively parallel screens utilised RNA-degrading CRISPR-Cas13 and 75,000 guide RNAs to precisely knock down lncRNAs without disrupting DNA regulatory elements, thereby leaving nearby protein-coding genes intact. The team, led by Neville Sanjana, identified 778 essential lncRNAs, with 46 showing universal essentiality and more than 700 with cell-specific roles. Essential lncRNAs were associated with cell cycle regulation and apoptosis pathways, often showing enriched expression in early developmental stages and specific tumours. Their findings were published yesterday in Nature Biotechnology.
• In an article published in Nature Communications, researchers in China introduce CHOOSER (Cas HOmlog Observing and SElf-processing scReening), an AI framework for alignment-free discovery of CRISPR-Cas systems with self-processing pre-crRNA capability using protein foundation models. The team reports that by using CHOOSER, they could identify 11 Casλ homologs, nearly doubling the known catalogue. They note that one homologue, EphcCasλ, was experimentally validated for self-processing pre-crRNA, DNA cleavage, and trans-cleavage, showing promise for CRISPR-based pathogen detection, and suggest that CHOOSER is an innovative approach for discovering CRISPR-Cas systems with specific functions and potential in gene-editing applications.
• Scientists in China and the United States have characterised the collateral activities of two Cas13 orthologs in mammalian cells, and found differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter. In their hands, LwaCas13a displayed greater activity that was has previously been reported in the literature, however this significant collateral RNA cleavage exerted only a modest impact on cell viability. They also found that collateral activity of LwaCas13a mildly impeded, but did not arrest, porcine embryo development. They suggest that their findings offer new insights into the implications of the collateral activity of Cas13 for clinical applications. Their findings were published in International Journal of Biological Macromolecules.
• Researchers at the National Human Genome Research Institute (NIH) in Bethesda report the development of a high-throughput method to synthesise and test the functionality of all possible in-frame and continuous deletions in a gene of interest, enabling rapid and unbiased determination of protein domain importance. Their method leverages a CRISPR library framework that is free from constraints of gRNA target site availability and efficacy. When applied to AcrIIA4, a phage-encoded anti-CRISPR protein that robustly inhibits SpCas9, they found that not all contacts with SpCas9 were required, despite previous structural characterisations revealing that AcrIIA4 physically occupies the DNA-binding interfaces of several SpCas9 domains. Of note, the team found that the AcrIIA4 loop that inserts into SpCas9's RuvC catalytic domain can be deleted. They conclude that their results show that AcrIIA4 inhibits SpCas9 primarily by blocking PAM binding and that its interaction with the SpCas9 catalytic domain is inessential. Their findings were published yesterday in PNAS.
• Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. In an article published yesterday in Nucleic Acids Research, a team in the United States reports that ssDNA with chemical modifications in 12-19% of internal bases, which they refer to as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells, CD34 + haematopoietic cells, T-cells and endothelial cells. They furthermore show therapeutically-relevant levels of gene correction in three clinically-relevant loci (CFTR, HBB and CCR5), which they report as being comparable to adeno-associated virus (AAV)-based templates.
• In an article published yesterday in Nucleic Acids Research, researchers in the United States report that the high efficiency of ABE8e - which is the most efficient adenine base editor reported to date - stems from TadA stable dimerisation in its TadA8e domain. They found that the dimers create strong interactions through R98 and R129 residues with Cas9's RuvC domain and DNA - a feature unique to ABE8e versus ABE7.10. In addition, they identified three key residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme's activity and stability, reinforcing the TadA8e dimer and improving the ABE8e's functionality.
• A team of researchers in Japan reports a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of the repeat expansion disorders, myotonic dystrophy type 1 (DM1). These orders are challenging to characterise and diagnose because of the variability and complexity of repeat lengths. The newly-introduced method, which uses in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. The team validated the robustness and accuracy of their method by quantitatively diagnosing DM1 in patient-isolated genomic DNA. Furthermore, they optimised a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. Their findings were published earlier this week in ACS Synthetic Biology.
• MicroRNA-21 or miR-21(a) is a known oncogene that is abundantly expressed in many cancer types including glioblastoma (GB), where it has been shown to promote disease progression, and lack of miR-21(a) has been demonstrated to reduce the tumorigenic potential. In the pursuit of a new way to treat GB, researchers in the United States and Netherlands propose a single adeno-associated virus (AAV) vector strategy targeting murine miR-21a using the ScCas9 ortholog guided by a single-guide RNA. They found that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumour site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered murine miR-21a levels in the brain, leading to reduced tumour growth and improved overall survival. Their findings were published in Molecular Therapy.

Industry

• Fate Therapeutics presented initial clinical data from the first patient treated in its FT819 Phase 1 trial for moderate-to-severe systemic lupus erythematosus (SLE) at the American College of Rheumatology Convergence held this week in Washington, D.C. The patient, a 27-year-old African American-Asian woman, received fludarabine-free conditioning followed by a single dose of FT819. The patient achieved clinical remission and low lupus disease activity state as of month 6 follow-up. The patient continues on-study, in clinical remission, and free of all immunosuppressive therapies as of a data cut-off date of November 11, 2024. FT819 is Fate's off-the-shelf, CD19-targeted, 1XX CAR T-cell product candidate comprised of CD8αβ+ T cells with a memory phenotype and high CXCR4 expression to promote tissue trafficking. For more details, see the official press release here. At the same meeting, Fate also presented initial Phase 1 clinical data for its off-the-shelf, ADR-armed CAR NK Cell Product Candidate, FT522. This data demonstrates a favourable safety profile, complete responses, and persistence of FT522 live cells in patients with relapsed or refractory B-cell lymphoma. Read more about that here.
• At the recent American Heart Association Scientific Sessions 2024, and in an article published last week in the New England Journal of Medicine, Intellia Therapeutics announced the first clinical evidence from its ongoing Phase 1 trial that Nexiguran Ziclumeran (nex-z, and formerly known as NTLA-2001) may favourably impact disease progression in transthyretin (ATTR) amyloidosis. Nex-z is an in vivo CRISPR-Cas9-based gene-editing therapy designed to treat ATTR by selectively reducing the levels of mutated TTR protein in the blood, through CRISPR-based inactivation of the TTR gene in liver cells. The candidate is jointly-developed with Regeneron and is currently being evaluated in two Phase 3 trials for ATTR with (ATTRv-PN) or without polyneuropathy. For further details see the official press release here.
• US-based company Korro Bio announced this week that it has obtained Australian HREC Approval and CTN Clearance to initiate Phase 1/2a clinical study (REWRITE) of KRRO-110 for alpha-1 antitrypsin deficiency. KRRO-110 is the first RNA-editing oligonucleotide product candidate from Korro’s proprietary RNA-editing platform, Oligonucleotide Promoted Editing of RNA (OPERA™). KRRO-110 is designed to co-opt an endogenous enzyme, adenosine deaminase acting on RNA (ADAR), to edit the “A” variant on SERPINA1 RNA, repair an amino acid codon, and restore secretion of normal AAT protein. See the official press release for more details.
• Earlier this week, Allogene Therapeutics announced pre-clinical data for ALLO-329, an investigational allogeneic TALEN-edited CD19/CD70 dual CAR T cell therapy candidate being evaluated as a treatment for autoimmune diseases. The data demonstrate that ALLO-329 induces deep, transient depletion of CD19+ B Cells and CD70+ T cells, and reduction in IgG and IgM without lymphodepletion in humanised murine models of lupus. ALLO-329 is the first CAR-T cell candidate designed to target both CD19+ B-cells and CD70+ activated T cells. Targeting of B cells has been demonstrated to induce durable, treatment-free remissions in patients with certain autoimmune diseases. CD70 is expressed in activated T cells, which have been implicated in immune responses, including in autoimmunity. Simultaneous elimination of CD70+ T cells is anticipated to increase the therapeutic benefit and expand the list of addressable indications. See the press release for further details.

Reviews

• Design, potential and limitations of conjugation-based antibacterial strategies. This review explores the design, potential, and limitations of conjugation-based antibacterial strategies, focusing on the recent advances in the delivery of CRISPR systems as antibacterial effectors.
• Navigating the intricate in-vivo journey of lipid nanoparticles tailored for the targeted delivery of RNA therapeutics: a quality-by-design approach. This review explores the structural and physicochemical characteristics of lipid nanoparticles (LNPs), their in vivo fates, and customisation for RNA therapeutics. The authors highlight the quality-by-design (QbD) approach for targeted delivery beyond the liver, focusing on biodistribution, immunogenicity, and toxicity. In addition, they discuss the current challenges and strategies associated with LNPs for in vivo RNA delivery, such as ensuring repeated-dose efficacy, safety, and tissue-specific gene delivery. Finally, the authors share insights into the current clinical applications in various classes of diseases as well as prospects of LNPs in RNA therapeutics.

Events

• CRISPR Genome Engineering and Therapy Symposium. This one-day event organised by Aarhus University Denmark and University of Copenhagen Denmark will features updates on the latest progress in CRISPR genome-editing technologies and therapeutic applications. See the programme and sign-up to attend here.

News from CRISPR Medicine News

• On Monday, we published an interview with Longzhu Cui, who is a Professor at Jichi Medical University in Japan. His team recently reported using phagemids for the delivery of CRISPR components to bacteriophage, selectively killing multi-drug resistant bacteria in vitro. Read the interview here.
• Tune Therapeutics recently received clinical trial application approval from the New Zealand Medicines and Medical Devices Safety Authority to initiate a Phase 1b clinical trial for TUNE-401, an investigational epigenetic silencing therapy designed to treat chronic Hepatitis B. At the AASLD conference held earlier this week, the company presented pre-clinical data demonstrating that TUNE-401 leads to robust, durable, and precise suppression of HBV DNA in both in vitro and in vivo models. Read more in our latest clinical update here.