Professional Education


  • Bachelor of Science, Virginia Polytechnic Institute & State University (2013)
  • Doctor of Philosophy, Washington University (2018)

Stanford Advisors


  • Mark Kay, Postdoctoral Faculty Sponsor

All Publications


  • Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice. Nature biotechnology Tsuji, S., Stephens, C. J., Bortolussi, G., Zhang, F., Baj, G., Jang, H., de Alencastro, G., Muro, A. F., Pekrun, K., Kay, M. A. 2022

    Abstract

    Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.

    View details for DOI 10.1038/s41587-022-01240-2

    View details for PubMedID 35393561

  • Lnc122-The miR122 Precursor Has an Independent Role as a Tumor Suppressor in Liver Jang, H., Chu, K., Zhang, F., Stephens, C., Kay, M. A. CELL PRESS. 2022: 514
  • Using Recombinant Adeno-Associated Viral Vectors for Long-Term Expression of a Hyperactive Human Factor IX Mutant in Hemophilic Mice and Comparison of AAV-LK03 and AAV-KP1 in Nonhuman Primates Pekrun, K., Stephens, C. J., Zhang, F., Kelly, L., Le Moan, N., Tarantal, A. F., Blouse, G. E., Kay, M. A. CELL PRESS. 2022: 330
  • Promoterless AAV Vectors with Homology Arms Can Integrate and Express from Transcriptionally Active Sites in Non-Targeted Loci Stephens, C. J., Pekrun, K., Xu, J., Tsuji, S., Jing, Y., Puzzo, F., Zhang, F., Kay, M. A. CELL PRESS. 2022: 391-392
  • Improving the In Vivo Gene Targeting Efficiency of Liver-Directed rAAV Vector Using the Nucleotide Analog Class of Ribonucleotide Reductase Inhibitors Tsuji, S., Stephens, C. J., Bortolussi, G., Pekrun, K., Zhang, F., de Alencastro, G., Baj, G., Muro, A. F., Kay, M. A. CELL PRESS. 2021: 163
  • Targeting Various Genomic Loci using AAV-GeneRide Results in Similar Genome Editing Efficiencies but May Affect Translation of the Chimeric mRNA Transcripts Stephens, C. J., Pekrun, K., Tsuji, S., Zhang, F., Kay, M. A. CELL PRESS. 2020: 455
  • Investigation of the Hepatocyte Population Amenable to Gene Targeting by rAAV-Mediated Homologous Recombination in Mice Tsuji, S., Stephens, C. J., Pekrun, K., Zhang, F., Zhang, A., Kay, M. A. CELL PRESS. 2020: 80
  • Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9 JOURNAL OF CONTROLLED RELEASE Stephens, C. J., Lauron, E. J., Kashentseva, E., Lu, Z., Yokoyama, W. M., Curiel, D. T. 2019; 298: 128-141

    Abstract

    Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.

    View details for DOI 10.1016/j.jconrel.2019.02.009

    View details for Web of Science ID 000460256900010

    View details for PubMedID 30771412

    View details for PubMedCentralID PMC6636336

  • Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9 GENE THERAPY Stephens, C. J., Kashentseva, E., Everett, W., Kaliberova, L., Curiel, D. T. 2018; 25 (2): 139-156

    Abstract

    Serum deficiency diseases such as alpha-1-antitrypsin deficiency are characterized by reduced function of serum proteins, caused by deleterious genetic mutations. These diseases are promising targets for genetic interventions. Gene therapies using viral vectors have been used to introduce correct copies of the disease-causing gene in preclinical and clinical studies. However, these studies highlighted that disease-alleviating gene expression is lost over time. Integration into a specific chromosomal site could provide lasting therapeutic expression to overcome this major limitation. Additionally, targeted integration could avoid detrimental mutagenesis associated with integrative vectors, such as tumorigenesis or functional gene perturbation. To test if adenoviral vectors can facilitate long-term gene expression through targeted integration, we somatically incorporated the human alpha-1-antitrypsin gene into the ROSA26 "safe harbor" locus in murine livers, using CRISPR/Cas9. We found adenoviral-mediated delivery of CRISPR/Cas9 achieved gene editing outcomes persisting over 200 days. Furthermore, gene knock-in maintained greater levels of the serum protein than provided by episomal expression. Importantly, our "knock-in" approach is generalizable to other serum proteins and supports in vivo cDNA replacement therapy to achieve stable gene expression.

    View details for DOI 10.1038/s41434-018-0003-1

    View details for Web of Science ID 000431216100007

    View details for PubMedID 29588497

    View details for PubMedCentralID PMC5919923