Compositions and methods for modifying a target nucleic acid
Inventors
Marson, Alexander • ROTH, Theodore Lee • Goodman, Daniel • Nguyen, David-Huy Nhu • Szoka, Jr., Francis C.
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Assignees
University of California San Diego UCSD
University of California, San Diego (UCSD)The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
The University of California, San Diego (UCSD) is a leading public research university located in La Jolla, California. Known for its innovative and interdisciplinary approach, UCSD offers a wide range of undergraduate, graduate, and professional programs across various fields. The university is committed to fostering a diverse and inclusive community, promoting sustainability, and driving social mobility through education, research, and public service. UCSD is recognized for its contributions to research and innovation, particularly in areas such as climate science, health innovation, and artificial intelligence.
Publication Number
US-12359179-B2
Publication Date
2025-07-15
Expiration Date
2039-12-12
Abstract
The disclosure provides compositions and methods for modifying a target nucleic acid. In some embodiments, a composition can include a targetable nuclease, a DNA-binding protein, and a donor template comprising a homology directed repair (HDR) template and one or more DNA-binding protein target sequences. In some embodiments, a composition can include a Cas protein, one or more single guide RNAs (sgRNAs), and an anionic polymer.
Core Innovation
The invention provides compositions and methods for modifying a target nucleic acid, wherein the composition comprises a targetable nuclease, a DNA-binding protein, and a donor template that includes a homology directed repair (HDR) template plus one or more DNA-binding protein target sequences. This configuration allows the DNA-binding protein to bind directly or indirectly to sequences on the donor template, facilitating shuttling of the HDR template to a cellular location proximal to the target nucleic acid, such as the nucleus, thus improving delivery and increasing knock-in efficiencies.
A key embodiment includes the use of RNA-guided nucleases, including CRISPR-Cas proteins, where both the targetable nuclease and DNA-binding protein may be RNA-guided nucleases. In some cases, the target gRNA is complementary to the target nucleic acid to direct cleavage, and a donor gRNA hybridizes to DNA-binding protein target sequences on the donor template to bring the template and nuclease complex together, promoting homologous recombination.
The background problem addressed is the limitations and complications associated with viral delivery vectors for gene therapy, such as insertional mutagenesis leading to leukemia and immune responses to viral vectors. Non-viral delivery methods like electroporation with CRISPR-Cas systems avoid these issues but face challenges including poor stability, RNP aggregation, high requirements for HDR template, and dose-dependent toxicity. The invention aims to resolve these challenges by improving the stability and delivery efficiency of genome editing components, thereby enhancing editing efficiency and cell viability in primary cells without requiring viral vectors.
Claims Coverage
The claims include multiple inventive features focusing on compositions and methods comprising a targetable nuclease, a DNA-binding protein, and a specially configured donor template.
Composition comprising a targetable nuclease, DNA-binding protein, and donor template with multiple PAMs and DNA-binding sequences
A composition for modifying a target nucleic acid comprising a targetable nuclease, a DNA-binding protein, and a donor template containing a homology directed repair (HDR) template, two or more protospacer adjacent motifs (PAMs), and two or more DNA-binding protein target sequences.
Use of RNA-guided nucleases as targetable nuclease and DNA-binding protein
Each of the targetable nuclease and the DNA-binding protein may be an RNA-guided nuclease, such as a Cas protein.
Inclusion of target guide RNA and donor guide RNA
The composition may further include a target guide RNA (gRNA) complementary to the target nucleic acid and a donor gRNA hybridizing to the DNA-binding protein target sequences on the donor template.
Complementarity of DNA-binding protein target sequences to donor gRNA
Each DNA-binding protein target sequence can be complementary to an equal length portion of the donor gRNA sequence to facilitate binding.
DNA-binding protein types and localization sequences
The DNA-binding protein may comprise transcription activator-like (TAL) effector DNA-binding protein or a zinc finger DNA-binding protein; the targetable nuclease and/or DNA-binding protein may be fused to a nuclear localization signal (NLS).
Targetable nuclease activity variations
The targetable nuclease may have nuclease activity or can lack nuclease activity.
Method of modifying target nucleic acid using the composition
A method comprises introducing the composition into a cell such that the HDR template is integrated into the target nucleic acid.
Donor template configuration with PAMs and DNA-binding sequences at termini
In the donor template, a first DNA-binding protein target sequence and a first PAM may be located at the 5′ terminus and a second DNA-binding protein target sequence and a second PAM at the 3′ terminus, with multiple options for PAM orientation relative to the DNA-binding target sequences.
The claims encompass compositions and methods for precise genome editing using a composition containing a targetable nuclease, DNA-binding protein, and a donor template with specific DNA-binding and PAM site configurations, including use of RNA-guided nucleases and guide RNAs, to improve targeted nucleic acid modifications with various configurations for enhanced efficiency and cellular localization.
Stated Advantages
Improved delivery of homology directed repair templates to the nucleus enhances genome targeting efficiency.
The system avoids complications associated with viral vectors, such as insertional mutagenesis and immune responses.
Addition of anionic polymers stabilizes Cas protein and RNP complexes, preventing aggregation and improving editing efficiency and cell viability.
The compositions and methods enable high efficiency large transgene knock-ins in diverse primary and stem cell types.
Capability to form lyophilized compositions for storage and later use without loss of editing activity.
Documented Applications
Non-viral genome editing of primary human T cells, including CD4+, CD8+, regulatory T cells, gamma-delta T cells, and natural killer (NK) cells.
Editing of primary human hematopoietic stem and progenitor cells (HSPCs) and iPS-derived stem cells using non-viral methods.
Correcting clinically relevant mutations or generating cells with exogenous genes such as chimeric antigen receptors (CARs) or T cell receptors (TCRs) for immunotherapy.
Multiplexed and bi-allelic gene editing for applications in adoptive cell therapies.
Use in various therapeutic cell engineering applications where viral delivery is unsuitable or undesirable.
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