Cindy Sandoval Espinoza

All Publications

• Characterizing and controlling CRISPR repair outcomes in nondividing human cells. Nature communications Ramadoss, G. N., Namaganda, S. J., Kumar, M. M., Hamilton, J. R., Sharma, R., Chow, K. G., Workley, L. A., Macklin, B. L., Sun, M., Ha, A. S., Liu, J., Fellmann, C., Watry, H. L., Dierks, P. H., Bose, R. S., Jin, J., Perez, B. S., Sandoval Espinoza, C. R., Matia, M. P., Lu, S. H., Judge, L. M., Shy, B. R., Nussenzweig, A., Adamson, B., Murthy, N., Doudna, J. A., Kampmann, M., Conklin, B. R. 2025; 16 (1): 9883

  Abstract

  Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.

  View details for DOI 10.1038/s41467-025-66058-3

  View details for PubMedID 41249169

• In vivo human T cell engineering with enveloped delivery vehicles NATURE BIOTECHNOLOGY Hamilton, J. R., Chen, E., Perez, B. S., Sandoval Espinoza, C. R., Kang, M., Trinidad, M., Ngo, W., Doudna, J. A. 2024; 42 (6): 860-871

  Abstract

  Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody-antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

  View details for DOI 10.1038/s41587-023-02085-z

  View details for Web of Science ID 001141658100003

  View details for PubMedID 38212493

  View details for PubMedCentralID PMC11236958

• CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells NUCLEIC ACIDS RESEARCH Lin-Shiao, E., Pfeifer, W. G., Shy, B. R., Saffari Doost, M., Chen, E., Vykunta, V. S., Hamilton, J. R., Stahl, E. C., Lopez, D. M., Sandoval Espinoza, C. R., Deyanov, A. E., Lew, R. J., Poirer, M. G., Marson, A., Castro, C. E., Doudna, J. A. 2022; 50 (3): 1256-1268

  Abstract

  DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

  View details for DOI 10.1093/nar/gkac049

  View details for Web of Science ID 000767881000010

  View details for PubMedID 35104875

  View details for PubMedCentralID PMC8860605

• Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering CELL REPORTS Hamilton, J. R., Tsuchida, C. A., Nguyen, D. N., Shy, B. R., McGarrigle, E., Espinoza, C., Carr, D., Blaeschke, F., Marson, A., Doudna, J. A. 2021; 35 (9): 109207

  Abstract

  As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.

  View details for DOI 10.1016/j.celrep.2021.109207

  View details for Web of Science ID 000658354200005

  View details for PubMedID 34077734

  View details for PubMedCentralID PMC8236216

• Engineering of monosized lipid-coated mesoporous silica nanoparticles for CRISPR delivery ACTA BIOMATERIALIA Noureddine, A., Maestas-Olguin, A., Saada, E. A., LaBauve, A. E., Agola, J. O., Baty, K. E., Howard, T., Sabo, J. K., Espinoza, C., Doudna, J. A., Schoeniger, J. S., Butler, K. S., Negrete, O. A., Brinker, C., Serda, R. E. 2020; 114: 358-368

  Abstract

  CRISPR gene editing technology is strategically foreseen to control diseases by correcting underlying aberrant genetic sequences. In order to overcome drawbacks associated with viral vectors, the establishment of an effective non-viral CRISPR delivery vehicle has become an important goal for nanomaterial scientists. Herein, we introduce a monosized lipid-coated mesoporous silica nanoparticle (LC-MSN) delivery vehicle that enables both loading of CRISPR components [145 µg ribonucleoprotein (RNP) or 40 µg plasmid/mg nanoparticles] and efficient release within cancer cells (70%). The RNP-loaded LC-MSN exhibited 10% gene editing in both in vitro reporter cancer cell lines and in an in vivo Ai9-tdTomato reporter mouse model. The structural and chemical versatility of the mesoporous silica core and lipid coating along with framework dissolution-assisted cargo delivery open new prospects towards safe CRISPR component delivery and enhanced gene editing. STATEMENT OF SIGNIFICANCE: After the discovery of CRISPR gene-correcting technology in bacteria. The translation of this technology to mammalian cells may change the face of cancer therapy within the next years. This was first made possible through the use of viral vectors; however, such systems limit the safe translation of CRISPR into clinics because its difficult preparation and immunogenicity. Therefore, biocompatible non-viral nanoparticulate systems are required to successfully deliver CRISPR into cancer cells. The present study presents the use of biomimetic lipid-coated mesoporous silica nanoparticles showing successful delivery of CRISPR ribonucleoprotein and plasmid into HeLa cervical and A549 lung cancer cells as well as successful gene editing in mice brain.

  View details for DOI 10.1016/j.actbio.2020.07.027

  View details for Web of Science ID 000567881300001

  View details for PubMedID 32702530