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Principal Investigator: JENNIFER A DOUDNA
Organization: UNIVERSITY OF CALIFORNIA BERKELEY
Fiscal Year: 2020
Award: $781,190
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY / ABSTRACT
CRISPR-Cas9 has demonstrated incredible potential to provide clinical benefit, but the challenge of delivery
currently hinders therapeutic use of genome editing in vivo. Use of viral vectors and lipid nanoparticles has
established the viability of in vivo genome editing, but these technologies have substantial drawbacks. Viral
vectors are immunogenic, difficult to manufacture, and have been associated with increased risks of off-target
editing. Lipid nanoparticles are unsuitable for systemic administration if targeting organs other than the liver. Ex
vivo therapies relying on autologous transplantation have shown the immense value in genetic manipulation of
immune cells, but the procedures remain risky, resource-intensive, and prohibitively expensive.
An ideal method to deliver therapeutic genome editing enzymes would be non-toxic, compatible with
intravenous administration, amenable to large-scale manufacture, and targeted to the cell type in need of
genetic correction. With all this in mind, we propose delivery of CRISPR-Cas9 in the form of an RNA-protein
(RNP) complex. Cas9 RNP has been shown to be safe and effective in vivo following local administration, and
we have established a strategy to enable cell type-specific delivery of Cas9 RNP tethered to a molecular
targeting agent (MTA) such as a receptor-binding ligand, antibody, or aptamer.
Our proposal aims to use MTA-tethered Cas9 RNP for targeted editing of T cells in vivo. We will rely on
established and novel MTAs to promote efficient and specific uptake of Cas9 RNP into T cells. Well-
characterized antibody MTAs will direct specific editing in human, mouse, and primate T cells. Novel aptamer
MTAs will be screened with a focus on cross-species reactivity to streamline the transition from pre-clinical to
clinical development. Because the Cas9 RNP has no inherent ability to cross cellular membranes, it will be
augmented with the ability to escape the endosome to avoid lysosomal degradation following MTA-induced
endocytosis. We have established a novel modular approach to functionalize Cas9 for endosomal escape,
facilitating re-optimization for specific cell types as needed.
In the UG3 phase, we will complete the following three aims: (1) Enable in vivo-compatible genome editing of
immune cells using targeted Cas9 RNP; (2) Identify robust molecular targeting agents for T cell-specific
editing; (3) Use targeted Cas9 RNP for in vivo genome editing of T cells. Following independent validation of
editing in mice, the UH3 phase will perform the following: (1) Scale up production of targeted Cas9 RNP for
large animal testing; (2) Validate targeted Cas9 RNP for in vivo genome editing in non-human primates.
The intersection of MTA-based cell targeting and the efficient endosomal escape of Cas9 RNP will generate a
versatile genome editing platform suitable for intravenous administration. Successful completion of the
proposed work will result in an engineered Cas9 RNP system that is safe, effective in vivo, readily
manufactured, and “plug & play” regarding its molecular targeting to multiple cell types of interest.
Terms: <Address><Animal Model><Animal Models and Related Studies><Animal Testing><Antibodies><Antibody Fragments><Autograft><Autologous Transplantation><Autotransplant><Binding><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell membrane><Cells><Cellular Membrane><Clinical><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><Cytoplasmic Membrane><Development><Disease><Disorder><Endocytosis><Endosomes><Engineering><Ensure><Enzyme Gene><Enzymes><Fruit><Genetic><High Throughput Assay><Homing><Human><Immune><Immunes><Immunoglobulin Fragments><Ligand Binding><Liver><M mulatta><M. mulatta><Macaca><Macaca mulatta><Macaque><Mediating><Methods><Mice><Mice Mammals><Mind><Modern Man><Molecular Interaction><Molecular Target><Murine><Mus><Non-Polyadenylated RNA><Organ><Peptides><Phase><Plasma Membrane><Play><Preparation><Primates><Primates Mammals><Procedures><Production><Property><Proteins><RNA><RNA Gene Products><Receptosomes><Research Resources><Resources><Rhesus Macaque><Rhesus Monkey><Ribonucleic Acid><Risk><Rodent><Rodentia><Rodents Mammals><Safety><Series><Specificity><Surface><System><T-Cells><T-Lymphocyte><Technology><Testing><Therapeutic><Therapeutic Uses><Validation><Viral Vector><Virus><Work><aptamer><autologous graft><autotransplantation><base><cell type><clinical development><cross reactivity><design><designing><developmental><dietary fruit><experiment><experimental research><experimental study><gene manipulation><gene-editing therapy><genetic manipulation><genetically manipulate><genetically perturb><genome editing><genome editing based therapy><genome editing therapy><genome editing treatment><genome editing-based therapeutics><genomic editing><hepatic body system><hepatic organ system><high throughput screening><humanized mice><humanized mouse><immunogenic><improved><in vitro testing><in vivo><interest><intravenous administration><lipid nanoparticle><model of animal><model organism><new technology><non-human primate><nonhuman primate><novel><novel technologies><plasmalemma><pre-clinical><preclinical><prevent><preventing><protein complex><receptor binding><receptor bound><recruit><scale up><screening><site targeted delivery><stem><targeted agent><targeted delivery><therapeutic editing><therapeutic genome editing><thymus derived lymphocyte><uptake>