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E2 - Other Nonviral Delivery (including lipid nanoparticles and exosomes)

1263: Novel Humanized Particles for Efficient Delivery of CRISPR and Other Gene Editors

Type: Poster Session

Poster Board Number: 1263
Presentation Details
Session Title: Thursday Posters: Other Nonviral Delivery

An urgent need exists for novel delivery modalities to fully realize the therapeutic potential of gene editing technologies including CRISPR. Recently described virus-like particles (VLPs) provide an alternative to more commonly used viral vectors such as AAV, offering advantages including delivery of a wide range of larger cargos (including ribonucleoproteins [RNPs]), transient cargo expression, and lack of potentially integrative DNA. However, VLPs still harbor multiple viral proteins, such as GAG, POL, and ENV, which may elicit immunogenicity and present potential challenges for manufacturing.
Here we describe the development and optimization of Enveloped Delivery for Generalized Entry (EDGE) and humanized-EDGE (h-EDGE) particles, novel delivery platforms offering the same advantages of VLPs but harboring fewer or no viral proteins, respectively.
EDGE particles do not contain viral GAG and POL proteins found in VLPs but instead utilize a compact human protein-derived architecture that we optimized to efficiently and non-covalently anchor cargos of interest to inner leaflet plasma membrane surfaces in EDGE producer cells. EDGE particles also harbor a viral ENV protein that enables high transduction efficiency and that distinguishes them from native extracellular vesicles. We show that EDGE particles can be used to deliver a wide range of gene editing cargos (CRISPR and non-CRISPR nucleases, base editors, and prime editors) and induce high frequency targeted edits into a wide variety of different cell types in culture (including primary human hepatocytes and primary human T cells). Importantly, we also show that EDGE particles can induce robust and efficient adenine base editing in vivo in mouse liver on a therapeutically relevant gene target (PCSK9).
We also describe the initial development and engineering of h-EDGE particles, which use the same non-covalent membrane anchoring technology in EDGE particles but utilize one of several human-derived ENV-alternative (ENV-alt) proteins instead of viral ENV proteins to achieve high transduction efficiencies. We show that h-EDGE particles pseudotyped with different ENV-alt proteins exhibit varied cell-type tropisms. These h-EDGE particles can deliver CRISPR gene editing nucleases and base editors into a broad range of therapeutically relevant primary human cells including HSCs, neurons, hepatocytes, and skeletal muscle, inducing highly efficient editing of as high as 50% or more. Because h-EDGE particles lack any viral proteins, they offer the potential for reduced immunogenicity and redosing in addition to a range of novel cell-type tropisms.
The EDGE and h-EDGE platforms provide new capabilities for delivering a wide range of diverse cargos to a variety of different cell types with potentially reduced immunogenicity and simplified profiles for manufacturing relative to VLPs. We anticipate that these new particles will be broadly useful for many research and therapeutics applications currently limited by the capabilities and characteristics of existing delivery modalities.

Peter K. Cabeceiras1,2,3,4,5, Noah Adams1, Tian Duo Wang2,3,5,6, Christopher Woodilla2,3,5, Caitlin Crandall1, Bevan Bsharah1, Xiaozhi Ren1, Yanjing Li1, Andrew Kane1, Jeffrey Lee1, Molly Riley1, Annie Elliott1, Vy Phan1, Carrie Ng7,8, Cosmin Mihai1, Jason Auer1, Casey A. Maguire7,8, Melissa Bonner1, J. Keith Joung2,3,4,5,6

1Nvelop Therapeutics, Cambridge, MA,2Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA,3Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA,4Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA,5Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA,6Department of Pathology, Harvard Medical School, Boston, MA,7Department of Neurology, Massachusetts General Hospital, Boston, MA,8Harvard Medical School, Boston, MA"

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