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Principal Investigator: David G. Keener
Organization: UNIV OF MASSACHUSETTS MED SCH WORCESTER
Fiscal Year: 2024
Award: $33,740
Funding agency: National Institute of Neurological Disorders and Stroke
PROJECT SUMMARY
Infants with Rett syndrome (Rett) are born with loss-of-function mutations in the gene encoding MeCP2, a global
regulator of gene expression. MeCP2 dysfunction in the brain severely affects neurons, leading to developmental
deficits of varying severity that manifest after 6 months of age. Current treatments can manage some symptoms,
but correcting MECP2 mutations would more effectively restore patients’ quality of life. CRISPR gene editing has
made this approach conceivable. Among CRISPR technologies, prime editing is the most flexible, utilizing an
RNA-guided Cas9 nuclease fused to reverse transcriptase to “search and replace” mutations in post-mitotic cells.
Thus, prime editing is a strong candidate for Rett treatment. Yet, prime editor has only been delivered to neurons
via lentivirus (not clinically relevant), and its editing efficiency in cells is low.
Previous work demonstrates that delivery of Cas9 mRNA encapsulated in lipid nanoparticles (LNP) is simple,
safe, and supports robust editing in mouse liver. LNP-encapsulated mRNA also delivers to brain, but delivery of
prime editor mRNA and efficiency of prime editing in neurons remains untested. In addition, chemically modifying
the guide RNA of other CRISPR systems can protect against nuclease-mediated degradation and improve gene
editing rates in cells. The Watts lab recently developed a method to synthesize chemically modified prime editing
guide RNA (pegRNA), something that was considered unfeasible due to the length of pegRNA (~150 nt). The
effect of pegRNA modification on prime editing efficiency has not yet been tested.
With support from Drs. Jonathan Watts (nucleic acid chemistry), Michael Green (Rett neurobiology), Erik
Sontheimer (prime editor biology), Scot Wolfe (gene regulation), and Athma Pai (bioinformatics), this project
seeks to establish and chemically optimize mRNA-based prime editors to correct MECP2 mutations and reverse
their phenotypes in neurons. Aim 1 will establish baseline effectiveness of mRNA-delivered prime editor (vs.
lentiviral) against the most common Rett mutation (a missense mutation) and two clinically severe nonsense
mutations in HEK cells expressing each mutant MeCP2, patient-derived induced pluripotent stem cells (iPSCs),
and iPSC-derived neurons. This Aim will also probe neurons with and without editing to understand the molecular
phenotypes of each MECP2 mutation and extent to which editing reverses them. Aim 2 will iterate on the Watts
lab’s pegRNA assembly method to optimize pegRNA yield and synthesis time, and identify editing-compatible
pegRNA modification patterns using in vitro and in cellulo assays. The effect of pegRNA modifications on MECP2
editing will be tested and optimized in HEK cells, iPSCs, and iPSC-derived neurons, as in Aim 1. Molecular
phenotypes of prime edited vs. unedited neurons will also be characterized as in Aim 1. This work will offer
insight into how MeCP2 mutants affect severity of Rett phenotypes in neurons and inform development of a
prime-editing platform to treat any form of Rett as well as other neurological disorders. The training provided
from this research will prepare the fellow for a productive career in the gene editing and neuro-therapeutics field.
Terms: <Affect><Age Months><Assay><Binding><Bio-Informatics><Bioassay><Bioinformatics><Biological Assay><Biology><Brain><Brain Nervous System><CRISPR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cells><Cerebroatrophic Hyperammonemia><Chemicals><Chemistry><Chimera Protein><Chimeric Proteins><Chromatin><Clinical><Closure by Ligation><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><DNA Integration><DNA Methylation><DNA delivery><DNA seq><DNA sequencing><DNAseq><Defect><Development><Dysfunction><EC 2.7.7.49><Effectiveness><Encapsulated><Encephalon><Engineering><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Functional disorder><Fusion Protein><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Genomic DNA><Guide RNA><Impairment><In Vitro><Induced pluripotent stem cell derived neurons><Infant><Interphase Cell><Length><Lentivirinae><Lentivirus><Ligation><Liver><Luciferase Immunologic><Luciferases><MeCP-2 protein><MeCP2><MeCP2 protein><Measures><Mediating><Messenger RNA><Methods><Methyl CpG Binding Protein 2><Methyl CpG binding protein MeCP2><Methyl-CpG binding protein 2><Methyl-CpG-Binding Protein 2><Methyl-DNA binding protein MECP2><Mice><Mice Mammals><Missense Mutation><Modification><Molecular><Molecular Interaction><Murine><Mus><Mutation><Nerve Cells><Nerve Unit><Nervous System Diseases><Nervous System Disorder><Neural Cell><Neurites><Neurobiology><Neurocyte><Neurodevelopmental Disorder><Neurologic><Neurologic Disorders><Neurological><Neurological Development Disorder><Neurological Disorders><Neuron from iPSC><Neuron from induced pluripotent stem cells><Neuronal Differentiation><Neurons><Non-dividing Cell><Nondividing Cell><Nonsense Mutation><Nuclear><Nucleic Acids><Patients><Pattern><Phenotype><Physiopathology><Primer Extension><Productivity><QOL><Quality of life><RNA Sequences><RNA Transcriptase><RNA chemical synthesis><RNA synthesis><RNA-Dependent DNA Polymerase><RNA-Directed DNA Polymerase><Reading Frames><Regulator Genes><Research><Resting Cell><Rett Disorder><Rett Syndrome><Reverse Transcriptase><Revertase><Severities><Symptoms><System><Technology><Testing><Therapeutic><Time><Training><Transcriptional Regulatory Elements><Transfection><Viral Vector><Work><career><chemical synthesis><curative intervention><curative therapeutic><curative therapy><curative treatments><deliver DNA><deliver mRNA><deliver messenger RNA><delivery system for mRNA><design><designing><developmental><entire genome><flexibility><flexible><flow cytophotometry><full genome><gDNA><gRNA><gene editing platform><gene editing system><gene editing technology><gene editing tools><gene-editing toolkit><genome mutation><genome sequencing><global gene expression><global transcription profile><hepatic body system><hepatic organ system><iPS><iPS neurons><iPSC><iPSC derived-neurons><iPSCs><immunogenic><improved><in vivo><induced pluripotent cell><induced pluripotent stem cell><induced pluripotent stem cell neurons><induced pluripotent stem cells derived from patients><induced pluripotent stem cells from patients><inducible pluripotent stem cell><insight><lipid based nanoparticle><lipid nanoparticle><loss of function mutation><mRNA><mRNA delivery><messenger RNA delivery><model organism><molecular phenotype><mutant><neurite growth><neurobiological><neurodevelopmental disease><neurological disease><neuron development><neuronal><neuronal development><neurons derived from induced pluripotent stem cells><non-sense mutation><novel><nuclease><pathophysiology><patient derived human iPS><patient derived human iPSC><patient derived human induced pluripotent stem cell><patient derived iPS><patient derived iPSC><patient derived induced pluripotent cells><patient derived induced pluripotent stem cells><patient-derived pluripotent stem cells><postmitotic><prime editing><prime editor><regulatory gene><technology platform><technology system><trans acting element><transcriptome><whole genome>