All Publications

  • High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition. Nature biotechnology Selvaraj, S., Feist, W. N., Viel, S., Vaidyanathan, S., Dudek, A. M., Gastou, M., Rockwood, S. J., Ekman, F. K., Oseghale, A. R., Xu, L., Pavel-Dinu, M., Luna, S. E., Cromer, M. K., Sayana, R., Gomez-Ospina, N., Porteus, M. H. 2023


    Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

    View details for DOI 10.1038/s41587-023-01888-4

    View details for PubMedID 37537500

    View details for PubMedCentralID 3694601

  • Establishing Multilayered Genetic Resistance to HIV-1 by Engineering Hematopoietic Stem and Progenitor Cells for B Cell Specific Secretion of Therapeutic Antibodies Feist, W., Luna, S., Ben-Efraim, K., Interrante, M., Amorin, N. A., Johnston, N., Dudek, A., Porteus, M. H. CELL PRESS. 2023: 115-116
  • A Simultaneous Knock-Out Knock-In Gene Editing Strategy in HSPCs Potently Inhibits R5-and X4-Tropic HIV Replication Dudek, A. M., Feist, W. N., Porteus, M. H. CELL PRESS. 2022: 230
  • GMEB2 is a Conserved Cellular AAV Restriction Factor That Inhibits Transduction of Human Stem Cells Dudek, A. M., Johnston, N. M., Vaidyanathan, S., Selvaraj, S., Porteus, M. H. CELL PRESS. 2021: 48-49
  • 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


    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

  • Answered and Unanswered Questions in Early-Stage Viral Vector Transduction Biology and Innate Primary Cell Toxicity for Ex-Vivo Gene Editing. Frontiers in immunology Dudek, A. M., Porteus, M. H. 2021; 12: 660302


    Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair. Many groups have used this strategy in development of therapies for blood and immune disorders such as sickle-cell anemia and severe-combined immunodeficiency. However, recent events have called into question the immunogenicity of AAV as a gene therapy vector and the safety profile dictated by the immune response to this vector. The target cells dictating this response and the molecular mechanisms dictating cellular response to AAV are poorly understood. Here, we will investigate the current known AAV capsid and genome interactions with cellular proteins during early stage vector transduction and how these interactions may influence innate cellular responses. We will discuss the current understanding of innate immune activation and DNA damage response to AAV, and the limitations of what is currently known. In particular, we will focus on pathway differences in cell line verses primary cells, with a focus on hematopoietic stem and progenitor cells (HSPCs) in the context of ex-vivo gene editing, and what we can learn from HSPC infection by other parvoviruses. Finally, we will discuss how innate immune and DNA damage response pathway activation in these highly sensitive stem cell populations may impact long-term engraftment and clinical outcomes as these gene-editing strategies move towards the clinic, with the aim to propose pathways relevant for improved hematopoietic stem cell survival and long-term engraftment after AAV-mediated genome editing.

    View details for DOI 10.3389/fimmu.2021.660302

    View details for PubMedID 34122418

  • Reply to "Efficient Nuclease-free HR by Clade F AAV Requires High MOIs with High Quality Vectors". Molecular therapy : the journal of the American Society of Gene Therapy Dudek, A. M., Porteus, M. H. 2019

    View details for DOI 10.1016/j.ymthe.2019.11.004

    View details for PubMedID 31735604

  • Genome-Wide CRISPR/Cas9 Screening Identifies GPR108 as a Highly Conserved AAV Entry Factor Dudek, A. M., Zinn, E., Pillay, S., Zengel, J., Carette, J. E., Vandenberghe, L. H. CELL PRESS. 2019: 313–14
  • GPR108 Is a Highly Conserved AAV Entry Factor. Molecular therapy : the journal of the American Society of Gene Therapy Dudek, A. M., Zabaleta, N. n., Zinn, E. n., Pillay, S. n., Zengel, J. n., Porter, C. n., Franceschini, J. S., Estelien, R. n., Carette, J. E., Zhou, G. L., Vandenberghe, L. H. 2019


    Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

    View details for DOI 10.1016/j.ymthe.2019.11.005

    View details for PubMedID 31784416

  • AAV6 Is Superior to Clade F AAVs in Stimulating Homologous Recombination-Based Genome Editing in Human HSPCs. Molecular therapy : the journal of the American Society of Gene Therapy Dudek, A. M., Porteus, M. H. 2019

    View details for DOI 10.1016/j.ymthe.2019.09.005

    View details for PubMedID 31537456

  • Identification and Characterization of an Alternate, AAVR Independent, AAV Entry Mechanism Using a Genome-Wide CRISPR/Cas9 Knock-Out Screen Dudek, A. M., Zinn, E. M., Pillay, S., Puschnik, A. S., Nagamine, C. M., Cheng, F., Qiu, J., Carette, J. E., Vandenberghe, L. H. CELL PRESS. 2018: 323
  • Delayed Onset and Altered Biodistribution of a Non-Canonical AAV Entry Pathway Dudek, A. M., Pillay, S., Puschnik, A. S., Nagamine, C. M., Carette, J. E., Vandenberghe, L. H. CELL PRESS. 2018: 188
  • An alternate route for adeno-associated virus entry independent of AAVR. Journal of virology Dudek, A. M., Pillay, S. n., Puschnik, A. S., Nagamine, C. M., Cheng, F. n., Qiu, J. n., Carette, J. E., Vandenberghe, L. H. 2018


    Determinants and mechanisms of cell attachment and entry steer the Adeno-Associated Virus (AAV) in its utility as a gene therapy vector. Thus far a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as in silico reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of multiplicity of infection. Viral overlay assays and rescue experiments in non-permisive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed in vivo, as AAVR knock out mice were equally permissive to transduction by AAVrh32.33 compared to wild type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both in vitro and in vivo, suggesting that some capsids may have a multi-modal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved amongst all primate AAVs except for those in the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of entry of AAV, a vector system of broad utility in gene therapy.Importance: Adeno-Associated Virus (AAV) is a non-pathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies of this vector.

    View details for PubMedID 29343568