Bio

Bio


I am a postdoctoral scholar working with Dr. Matthew Porteus. Gene therapy has been my primary research interest during my doctoral and postdoctoral training. As a doctoral student, I studied the intracellular transport of non-viral gene delivery vectors to optimize delivery. I joined the Porteus lab to further my interest in gene therapy by applying CRISPR/Cas9 based genome editing for monogenic diseases. As a postdoctoral scholar, I have been working on using CRISPR/Cas9 technology to develop an autologous gene corrected airway stem cell therapy to treat cystic fibrosis.

Academic Appointments


Honors & Awards


  • Postdoctoral Fellowship Award, Cystic Fibrosis Foundation (May 2019-March 2021)
  • K99/R00 Pathway to Independence Award, National Institutes of Health (March 2021 - Present)
  • School of Medicine Dean's Fellowship, Stanford University (January - December 2017)

Publications

All Publications


  • Correction of Recessive Dystrophic Epidermolysis Bullosa by homology-directed repair-mediated genome editing. Molecular therapy : the journal of the American Society of Gene Therapy Bonafont, J. n., Mencía, A. n., Chacón-Solano, E. n., Srifa, W. n., Vaidyanathan, S. n., Romano, R. n., Garcia, M. n., Hervás-Salcedo, R. n., Ugalde, L. n., Duarte, B. n., Porteus, M. H., Del Rio, M. n., Larcher, F. n., Murillas, R. n. 2021

    Abstract

    Genome editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa is a group of rare genetic disorders characterized by extreme skin fragility. The Recessive Dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. Here, we report a gene editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR/Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAV). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof-of-concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.

    View details for DOI 10.1016/j.ymthe.2021.02.019

    View details for PubMedID 33609734

  • Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus. Molecular therapy : the journal of the American Society of Gene Therapy Vaidyanathan, S. n., Baik, R. n., Chen, L. n., Bravo, D. T., Suarez, C. J., Abazari, S. M., Salahudeen, A. A., Dudek, A. M., Teran, C. A., Davis, T. H., Lee, C. M., Bao, G. n., Randell, S. H., Artandi, S. E., Wine, J. J., Kuo, C. J., Desai, T. J., Nayak, J. V., Sellers, Z. M., Porteus, M. H. 2021

    Abstract

    Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

    View details for DOI 10.1016/j.ymthe.2021.03.023

    View details for PubMedID 33794364

  • Insertion of the CFTR cDNA in the Endogenous Locus in Airway Stem Cells Using CRISPR/Cas9 Restores CFTR Function to Wild-Type Levels in Differentiated Epithelia Vaidyanathan, S., Sellers, Z. M., Bravo, D. T., Le, W., Randell, S. H., Desai, T. J., Kuo, C. J., Nayak, J. V., Porteus, M. H. CELL PRESS. 2020: 569?70
  • Precise COL7A1 Gene Correction in Primary Patient Cells as a Therapeutic Option for Epidermolysis Bullosa Bonafont, J., Mencia, A., Srifa, W., Vaidyanathan, S., Romano, R., Garcia, M., Jose Escamez, M., Duarte, B., Porteus, M., Larcher, F., Del Rio, M., Murillas, R. CELL PRESS. 2020: 325?26
  • Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination CELL STEM CELL Martin, R. M., Ikeda, K., Cromer, M., Uchida, N., Nishimura, T., Romano, R., Tong, A. J., Lemgart, V. T., Camarena, J., Pavel-Dinu, M., Sindhu, C., Wiebking, V., Vaidyanathan, S., Dever, D. P., Bak, R. O., Laustsen, A., Lesch, B. J., Jakobsen, M. R., Sebastiano, V., Nakauchi, H., Porteus, M. H. 2019; 24 (5): 821-+
  • High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia. Cell stem cell Vaidyanathan, S. n., Salahudeen, A. A., Sellers, Z. M., Bravo, D. T., Choi, S. S., Batish, A. n., Le, W. n., Baik, R. n., de la O, S. n., Kaushik, M. P., Galper, N. n., Lee, C. M., Teran, C. A., Yoo, J. H., Bao, G. n., Chang, E. H., Patel, Z. M., Hwang, P. H., Wine, J. J., Milla, C. E., Desai, T. J., Nayak, J. V., Kuo, C. J., Porteus, M. H. 2019

    Abstract

    Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ?F508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

    View details for DOI 10.1016/j.stem.2019.11.002

    View details for PubMedID 31839569

  • Compositional Heterogeneity in Lumbar Vertebral Trabecular Bone as a Function of Disease and Treatment Colon-Bernal, I., Yang, P., Ahn, T., Duong, L., Pennypacker, B., Cauble, M., Vaidyanathan, S., Kozloff, K., Orr, B., Holl, M. WILEY. 2018: 294
  • Tailoring dendrimer conjugates for biomedical applications: the impact of altering hydrophobicity JOURNAL OF NANOPARTICLE RESEARCH Holl, M., Dougherty, C. A., Vaidyanathan, S. 2018; 20 (10)
  • Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Molecular therapy. Nucleic acids Vaidyanathan, S., Azizian, K. T., Haque, A. K., Henderson, J. M., Hendel, A., Shore, S., Antony, J. S., Hogrefe, R. I., Kormann, M. S., Porteus, M. H., McCaffrey, A. P. 2018; 12: 530?42

    Abstract

    The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average± SD= 79%± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both invitro and invivo.

    View details for PubMedID 30195789

  • Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells. Molecular therapy : the journal of the American Society of Gene Therapy Cromer, M. K., Vaidyanathan, S. n., Ryan, D. E., Curry, B. n., Lucas, A. B., Camarena, J. n., Kaushik, M. n., Hay, S. R., Martin, R. M., Steinfeld, I. n., Bak, R. O., Dever, D. P., Hendel, A. n., Bruhn, L. n., Porteus, M. H. 2018

    Abstract

    Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

    View details for PubMedID 30005866

  • Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting Molecular Therapy Nucleic Acids Charlesworth, C. T., Camarena, J., Cromer, M. K., Vaidyanathan, S., Bak, R. O., Carte, J. M., Potter, J., Dever, D. P., Porteus, M. H. 2018; 12: 89-104
  • Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. Molecular therapy. Nucleic acids Charlesworth, C. T., Camarena, J. n., Cromer, M. K., Vaidyanathan, S. n., Bak, R. O., Carte, J. M., Potter, J. n., Dever, D. P., Porteus, M. H. 2018; 12: 89?104

    Abstract

    Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the ?-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of ?-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

    View details for PubMedID 30195800

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