2023 Cold Spring Harbor Laboratory Genome Engineering: CRISPR Frontiers

Raoul Martin, Craig Soares, Boris Korablev, Grace Lee, Madhurya Sekhar, Lenaig Defachelles, Shannon McCawley, Vihasi Jani, Jean Chan, Weng-In Leong, Rina Mepani, Benjamin G Gowen, Mark W Knuth, Aaron J Cantor, Peter Cameron

Delivering CRISPR machinery to specific cells in vivo remains a major challenge for therapeutic gene editing. Directed delivery of a Cas9 Ribonucleoprotein (RNP) complex offers many advantages: it avoids risks associated with continuous nuclease expression or integration of genetic elements, obviates cargo capacity limitations inherent to viral vectors, and offers a cost-effective manufacturing workflow wherein a single preparation of endonuclease can be used with multiple sgRNAs. Unmodified Cas9 RNPs, however, lack robust mechanisms for cell penetration—this activity must be integrated through protein engineering. Here, we describe the development of a highly cell-penetrant Cas9 RNP. To engineer this activity, we used a small protein scaffold linked to Cas9 to display cell penetrating peptides (CPPs) at a high local concentration. The CPPs were attached via cysteine-disulfide linkages under the rationale that release of the CPPs in the cytosol would prevent interference with downstream editing function. Parallel screening of CPPs and small, thermostable protein scaffolds converged to a TIM barrel variant displaying high-performing CPPs via a specific configuration of 4 engineered cysteines. Notably, this engineered RNP edited human primary T cells at up to 70% efficiency upon simple co-incubation for 1 hour in standard media (with no added delivery agents). This work highlights the potential for engineering robust cell-penetrating activity directly into CRISPR RNPs and establishes a foundation for future improvements, including integration of cell-targeting activity and delivery to specific cell types in vivo.

Inaugural In Vivo Engineering of Therapeutic Cells Summit (July 14, 2022)

- Developing TAGE (Targeted Active Gene Editors), a new class of in vivo cell-targeting CRISPR RNP-based biologics

- Applying TAGE to expand the target space in immuno-oncology (IO), as well as for monogenic diseases therapies

- IO TAGE, targeting selected immune cells, reprogram the tumor microenvironment to facilitate a systemic anti-tumor response

2022 Keystone Symposia: Precision Genome Engineering - April 27, 2022

Spencer Wei, Ashil Bans, Pierre Boivin, WengIn Leong, Jean Chan, Benjamin Gowen, Shannon McCawley Rina Mepani, Angela Cabral, Mark Knuth, Aaron Cantor, Eric Estrin, Akshay Tambe, Kory Melton, Hari Jayaram, Peter Cameron, Mary Haak-Frendscho, Mary Janatpour

Spotlight Therapeutics is developing RNP-based biologics to enable gene editing of selected cell types in vivo. This new class of modular RNPs, called TAGE (Targeted Active Gene Editors), bypass viral and nanoparticle packaged delivery methods. TAGE are CRISPR nuclease effectors engineered with a targeting antibody (Ab) moiety and cell penetrating peptide (CPP) domains. TAGE optimization is achieved by exploiting synergistic interactions between Ab and CPP domains to achieve efficient cell targeting, transmembrane trafficking and nuclear localization. We are applying TAGE technology to address both monogenic diseases and to develop novel therapeutic strategies that expand the target space in immuno-oncology (IO). Here we show margination of prototype TAGE to the immune compartment of tumors following local injection. Subsequent CRISPR Cas9-mediated knockout of otherwise ‘difficult-to-drug’ IO gene targets in the selected cell types modulate the tumor microenvironment to potentiate a systemic anti-tumor response. This differentiated approach is designed to engage both the innate and adaptive immune system to prime a new T cell response in combination with immune checkpoint blockade. The ability to selectively edit cells in vivo with a modular biologic modality has the potential to greatly expand therapeutic applications and increase patient access to gene editing medicines.

2021 ASGCT Annual Conference Poster 412 - May 11, 2021

Rina Mepani(1*), Pawan Kumar Shahi(2,4*), Aaron J. Cantor(1), Aarushi Grover(1), Nicholas Albanese(1), Mary J. Janatpour(1), Mary Haak-Frendscho(1), Hariharan Jayaram(1), Krishanu Saha(3,4) and Bikash R. Pattnaik(2,4)

(1)Spotlight Therapeutics, 26118 Research Place, Hayward, CA 94545, (2)Department of Pediatrics, University of Wisconsin-Madison, (3)Wisconsin Institute for Discovery, University of Wisconsin-Madison, (4)McPherson Eye Research Institute, University of Wisconsin-Madison, (*)These authors contributed equally.

Despite remarkable advances, delivery remains the most significant hurdle to realizing the full therapeutic potential of genome editing. To enable in vivo cell-selective delivery of gene editing molecules safely and efficiently, we developed a new class of biologics called Targeted Active Gene Editors (TAGE). TAGE are modular, programmable ribonucleoproteins that target selected cell types and edit specific genes. Here, we demonstrate preclinical proof of principle with in vivo retinal pigment epithelium (RPE) cell editing following a single subretinal TAGE administration. Using the same ocular-targeted scaffold, we evaluated whether the TAGE could mediate a functional effect in an RPE functional model. These studies, bypassing the complexities and safety concerns associated with viral and nanoparticle delivery vehicles, demonstrate the broad potential of in situ TAGE delivery for gene editing.

2021 ASGCT Annual Conference Workshop: Moving Genome Editing to the Clinic: From Technology to Therapeutics - May 10, 2021

Spotlight Therapeutics is developing programmable RNPs to edit selected cell types in vivo. This new class of modular RNPs, called TAGE (Targeted Active Gene Editors), are designed to address the limitations and challenges of viral and nanoparticle delivery methods. TAGE are engineered for a “hit-and-run” gene editing mechanism to minimize unintended gene silencing and increase their overall safety profile. TAGE optimization is achieved by combining a cell targeting antibody with CPPs, identified from our proprietary TAGE library screen, and linked to gRNA-loaded nuclease. The ability to selectively edit cells in vivo has the potential to greatly expand both indications and patient access to effective gene editing therapies.

Cold Spring Harbor meeting on Genome Engineering: CRISPR Frontiers, Therapeutics, session 8, oral presentation – Aug 21, 2020

Akshay Tambe, Rina J Mepani, Aaron J Cantor, Sruja S Iyer, Angelica F Castaneda, Hariharan Jayaram

Spotlight Therapeutics, Hayward, CA

Editing genomes with CRISPR-Cas proteins has the potential to revolutionize the way human genetic diseases are treated. Although remarkable progress has been made using these nucleases to modify a genome in a defined manner, delivering Cas / guide RNA complexes to selected cells remains a significant challenge.

To address this, we developed a high-throughput screen to identify Cas fusion proteins that can selectively internalize and localize to the nucleus of primary cells. This workflow does not require engineering of reporters into the target cells, thereby enabling the use of primary cells and a wide variety of cell types. First, we developed a streamlined and scalable technique to prepare large libraries of RNA-barcoded Cas9 complexes, which pairs unique sequence identifiers on the guide RNA with a library of Cas9 protein variants. Then we identified cell-internalizing variants by co-incubating the RNA-barcoded protein complexes with cells of interest, isolating the nuclear subcompartment, and sequencing the barcodes within that fraction. Quantitation of the internalized barcodes allows us to measure the uptake dynamics of multiple Cas9 variants simultaneously.

To demonstrate feasibility, we prepared a library of approximately five thousand Cas9 variants, each bearing a unique cell penetrating peptide and its identifying guide RNA barcode. We then used our high-throughput sequencing assay to quantitatively rank each cell penetrating peptide’s capacity to deliver the Cas9 payload to the nucleus of primary mouse fibroblasts. As an internal validation of the method, the library included sequences that have been previously shown to improve Cas9 internalization, and as expected, the screen recovered the positive control peptides. We also identified a number of previously uncharacterized peptides that enhance Cas9 internalization. Global analysis of the chemical properties of the peptide library revealed chemical signatures that substantially improve Cas9 internalization.

Taken together, our methods serve as a proof-of-principle for a pooled screen for CRISPR-Cas delivery. It can be easily expanded to investigate larger and more diverse libraries of Cas variants, as well as other cell types of interest. Additionally, our approach to preparing libraries of barcoded CRISPR-Cas proteins is likely to be widely applicable to other protein engineering problems in the field.

2020 ASGCT Annual Conference, Gene Regulation and Delivery Technologies session, oral presentation – May 15, 2020

ASGCT recorded webinar Link

Rina Mepani(*), Kathryn Logronio(*), Aaron J. Cantor, Sruja Iyer, Nicholas Albanese, Benjamin G. Gowen, Spencer Wei, Mary J. Janatpour, Mary Haak-Frendscho, Hariharan Jayaram

Spotlight Therapeutics, 26118 Research Place, Hayward, CA 94545

(*)These authors contributed equally

Despite remarkable advances in gene editing, the full therapeutic potential of this promising modality is hampered by delivery. To address the multiple limitations of viral- and nanoparticle-mediated delivery for gene editing, we are developing a new class of biologics called Targeted Active Gene Editors (TAGE). TAGE are modular, programmable ribonucleoproteins (RNPs) comprising a cell-targeting domain linked to a Cas protein loaded with sgRNA. These dual function TAGE are uniquely designed for genome editing of selected cell types in situ. Here, we demonstrate preclinical proof of concept by editing hematopoietic stem and progenitor cells (HSPCs). Gene editing of human HSPCs was achieved directly using naked RNP, bypassing the need for an exogenous delivery vehicle. Moreover, direct intraosseous injection of TAGE in the Ai9 reporter mouse resulted in editing of bone marrow cells, including long-term progenitor HSCs (LSK CD150+ CD34-).