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C4 - Targeted Gene Insertion (integrase mediated insertion -targeted or safe harbor)

195: Programmable Genomic Integration in Induced Pluripotent Stem Cells and Hematopoietic Stem and Progenitor Cells

Type: Oral Abstract Session

Presentation Details
Session Title: Targeted Gene Insertion

CRISPR/Cas9-based technologies have revolutionized basic and applied biomedical research in the past decade. Current targeted gene insertion approaches require DNA double-strand breaks (DSBs), risking translocations, and rely on DNA repair pathways (e.g., homology-directed repair, HDR) that are inactive in quiescent or terminally differentiated cells. Programmable and multiplexed genome integration of multi-kilobase (kb) DNA cargo independent of HDR is still challenging. Here, we present a proof-of-concept (PoC) evaluation of Tome Biosciences’ proprietary technology, integrase-mediated programmable genomic integration (I-PGI), on induced pluripotent stem cells (iPSCs) and CD34+ hematopoietic stem cells (HSCs). I-PGI combines CRISPR-mediated genome editing and site-specific integrases, enabling efficient and targeted genomic integration without DSBs.
Two unique features of iPSCs, unlimited expansion and the ability to differentiate into almost all cell types, provide an unprecedented opportunity for cell therapy. The current FDA-approved autologous cell therapies are costly, labor-intensive, time-consuming to manufacture, and carry a risk of oncogenic transformation due to random integration using lentivirus. Allogeneic iPSC-derived cell products, which can be manufactured on a large scale and used “off-the-shelf”, hold promise to overcome these limitations. First, we leveraged attachment site-containing guide RNAs (atgRNAs), Cas9 nickase, and reverse transcriptase to place integrase-recognizing landing sites (beacons) at the B2M locus and achieved over 90% beacon placement (BP) efficiency. We followed by integrating a DNA cargo of 5.2kb into the placed beacon and achieved up to 46% efficiency. We then tested a template of 31kb and successfully reached 6% integration. To assess multiplex integration, we simultaneously placed orthogonal beacons at four different genetic loci and integrated 4 corresponding templates into these beacons. We observed integration of 1.4%, 6.8%, 8.1% and 10.2% respectively with DNA templates ranging in size from 4 to 6kb.
HSC transplantation has been a curative therapy for many hematological disorders and malignancies in the last few decades, and the field is further energized by the recent approval of Casgevy. However, durable gene replacement requires editing of the quiescent long-term (LT)-HSC population, which is limited by HDR-mediated approaches. In donor-derived CD34+ HSCs, we achieved BP efficiencies of over 80% at the B2M locus and over 30% I-PGI efficiencies. To determine if I-PGI can engineer LT-HSCs, we isolated the primitive HSC sub-population and demonstrated that PGI efficiencies correlated with the bulk engineered HSC population. We are currently evaluating if the I-PGI engineered HSC successfully engraft and persist in vivo.
In summary, our PoC study demonstrated efficient, DSB-free engineering of iPSCs and HSPCs by I-PGI technology. This novel approach gives us the advantage of speed, precision, and specificity for next generation cell therapies.

Minggang Fang, Ravindra Amunugama, Kaivalya Molugu, Marie-Joe Kimaz, Lauren Herchenroder, Adam Zieba, Jie Wang, Wei Wang, Kangni Zheng, Jason Zhang, Devin Harrison, Sandeep Kumar, Daniel O'Connell, Chong Luo, Jonathan Finn

Tome Biosciences, Watertown, MA"

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