Date and time: January. 8^th 2PM

In-person location: Singleton Auditorium (46-2005)

On-Zoom https://mit.zoom.us/j/99421924894

Title: RNA and DNA editing for genetic disorders

Abstract:  The prospect of genetic editing is an appealing therapeutic option for many inherited diseases. However, precise genetic manipulation remains limited by challenges in editing efficiency, cargo size constraints, delivery specificity, and off-target effects. We developed a suite of technologies that aim to address these limitations through advances in RNA and DNA editing and non-viral delivery.  Chapter 2 starts from work on CRISPR-Cas9 and prime-editing. We develop PASTE (Programmable Addition via Site-specific Targeting Elements), which leverages existing prime editing strategies to install “beacon” sites for serine integrases such as Bxb1 to insert a larger payload. This system enables targeted insertion of sequences up to ~36 kilobases without relying on DNA double strand break repair pathways Through metagenomic discovery and subsequent engineering, we achieved efficient integration across human cell lines, primary T cells, and non-dividing hepatocytes. PASTE demonstrates editing efficiencies comparable to or exceeding HDR and NHEJ-based methods, with fewer adverse off-target events. In Chapter 3, we start from the RNA nuclease Cas7-11, discovered in a transcriptional cassette flanked by several other proteins and RNA features. Through structural analysis, we describe a Cas7-11-Csx29-Csx30 “Craspase” system, which we functionally characterized, demonstrating the first instance of an RNA-guided protease. We repurpose this system for programmable RNA sensing in mammalian cells, allowing for the creation of a sensitive molecular reporter. Continuing our work with Cas7-11 in Chapter 4, we next developed PRECISE (Programmable RNA Editing & Cleavage for Insertion, Substitution, and Erasure), which enables versatile RNA editing via 5′and 3′ trans-splicing assisted by RNA-cleaving ribozymes and RNA stability elements. We demonstrated edits spanning all 12 substitutions, plus deletions and insertions from 1 to 5 thousand nucleotides across nearly 20 endogenous genes, with no detectable off-targets. We demonstrate therapeutic application through editing of transcripts causative of Rett Syndrome and Huntington’s disease.  Recognizing that many DNA and RNA tools show promise in the laboratory but are complicated to deliver in vivo or package for a therapeutic, in Chapter 5 we shift focus to nucleic acid delivery. A promising modality for in vivo delivery of gene therapies is lipid nanoparticles (LNP), but LNPs are limited by bias towards liver transduction, largely due to interactions between LNP-adsorbed sera proteins and LDLR-expressing hepatocytes. To address this, we engineered "dead ApoE" (dApoE) apolipoprotein mutants with five receptor-binding domain substitutions to disrupt ApoE-LDLR interactions while preserving LNP binding. We showed that coating LNPs with dApoE, when combined with existing retargeting antibodies (e.g. CD5 for T cells), provides a modular framework to reduce hepatic transduction by 90% while achieving equivalent levels of tissue specific delivery in brain, T cells and lung. We use this strategy to generate ~10% functional CAR+ T cells with preserved cytotoxicity in vivo, demonstrating therapeutic viability. Taken together, these technologies represent an effort to push forward our capabilities for genetic medicine: RNA-level editing deliverable via AAV; large-payload genome integration; RNA targeting and sensing capabilities; and tissue-specific delivery without hepatic off-targets. This integrated toolkit expands the scope of gene therapy for treating genetic diseases, cancer, neurological disorders, and immune dysfunction.