Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver

Sago, Cory D.; Lokugamage, Melissa P.; Loughrey, David; Lindsay, Kevin E.; Hincapie, Robert; Krupczak, Brandon R.; Kalathoor, Sujay; Sato, Manaka; Echeverri, Elisa Schrader; Fitzgerald, Jordan P.; Gan, Zubao; Gamboa, Lena; Paunovska, Kalina; Sanhueza, Carlos; Hatit, Marine Z. C.; Finn, M. G.; Santangelo, Philip J.; Dahlman, James E.

doi:10.1038/s41551-022-00847-9

Cited by 44 publications

(43 citation statements)

References 62 publications

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“…Although the effects of deletion mutagenesis on cell-specific splicing can be unpredictable, recently developed machine learning algorithms may help facilitate rational design of smaller SLED vectors 75 , 76 . Drug-inducible approaches to regulate splicing 77 – 79 , inclusion of miRNA target sites 80 , 81 , and optimizations based on synthetic introns 60 may enable further control of SLED-based constructs.…”

Section: Discussionmentioning

confidence: 99%

Cell-specific regulation of gene expression using splicing-dependent frameshifting

et al. 2022

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Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.

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Section: Discussionmentioning

confidence: 99%

Cell-specific regulation of gene expression using splicing-dependent frameshifting

et al. 2022

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“…While the effects of deletion mutagenesis on cell-specific splicing can be unpredictable, recently developed machine learning algorithms may help facilitate rational design of smaller SLED vectors 69,70 . Drug-inducible approaches to regulate splicing [71][72][73] and the inclusion of miRNA target sites 74,75 may enable further control of SLED-based constructs.…”

Section: Discussionmentioning

confidence: 99%

Cell-specific regulation of gene expression using splicing-dependent frameshifting

Ling

Bygrave

Santiago

et al. 2022

Preprint

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Precise and reliable gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA-Seq datasets, and filtered by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED vectors can leverage the strong expression of constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. We generated SLED vectors to selectively target all neurons, photoreceptors, or excitatory neurons, and demonstrated that specificity was retained in vivo when delivered using AAV vectors. We further demonstrated the utility of SLED by creating what would otherwise be unobtainable research tools, specifically a GluA2 flip/flop reporter and a dual excitatory/inhibitory neuronal calcium indicator. Finally, we show that SLED vectors can rescue photoreceptor degeneration in Prph2rds/rds mice, and selectively induce apoptosis in SF3B1 mutant cancer cells with an oncolytic vector. The flexibility of SLED technology enables new avenues for basic and translational research.

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“…To target non-hepatocytes, scientists have used three approaches 14 . The first is to pre-treat animals with systems that overwhelm the liver 15 or reduce drug activity 16 in specific cell types, thereby shifting tropism. However, it remains unclear whether this multistep strategy has clinical relevance.…”

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confidence: 99%

Piperazine-derived lipid nanoparticles deliver mRNA to immune cells in vivo

Hatit

Zhao

et al. 2022

Nat Commun

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In humans, lipid nanoparticles (LNPs) have safely delivered therapeutic RNA to hepatocytes after systemic administration and to antigen-presenting cells after intramuscular injection. However, systemic RNA delivery to non-hepatocytes remains challenging, especially without targeting ligands such as antibodies, peptides, or aptamers. Here we report that piperazine-containing ionizable lipids (Pi-Lipids) preferentially deliver mRNA to immune cells in vivo without targeting ligands. After synthesizing and characterizing Pi-Lipids, we use high-throughput DNA barcoding to quantify how 65 chemically distinct LNPs functionally delivered mRNA (i.e., mRNA translated into functional, gene-editing protein) in 14 cell types directly in vivo. By analyzing the relationships between lipid structure and cellular targeting, we identify lipid traits that increase delivery in vivo. In addition, we characterize Pi-A10, an LNP that preferentially delivers mRNA to the liver and splenic immune cells at the clinically relevant dose of 0.3 mg/kg. These data demonstrate that high-throughput in vivo studies can identify nanoparticles with natural non-hepatocyte tropism and support the hypothesis that lipids with bioactive small-molecule motifs can deliver mRNA in vivo.

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Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver

Cited by 44 publications

References 62 publications

Cell-specific regulation of gene expression using splicing-dependent frameshifting

Cell-specific regulation of gene expression using splicing-dependent frameshifting

Cell-specific regulation of gene expression using splicing-dependent frameshifting

Piperazine-derived lipid nanoparticles deliver mRNA to immune cells in vivo

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