Academic Appointments

Professional Education

  • PhD, Yale University, Genetics (2014)
  • Postdoctoral Fellowship, Harvard Medical School, Genetics (2016)

All Publications

  • Gene replacement of alpha-globin with beta-globin restores hemoglobin balance in beta-thalassemia-derived hematopoietic stem and progenitor cells. Nature medicine Cromer, M. K., Camarena, J., Martin, R. M., Lesch, B. J., Vakulskas, C. A., Bode, N. M., Kurgan, G., Collingwood, M. A., Rettig, G. R., Behlke, M. A., Lemgart, V. T., Zhang, Y., Goyal, A., Zhao, F., Ponce, E., Srifa, W., Bak, R. O., Uchida, N., Majeti, R., Sheehan, V. A., Tisdale, J. F., Dever, D. P., Porteus, M. H. 2021


    beta-Thalassemia pathology is due not only to loss of beta-globin (HBB), but also to erythrotoxic accumulation and aggregation of the beta-globin-binding partner, alpha-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in beta-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize beta-globin:alpha-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing beta-thalassemia.

    View details for DOI 10.1038/s41591-021-01284-y

    View details for PubMedID 33737751

  • Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex. Molecular therapy : the journal of the American Society of Gene Therapy de Alencastro, G. n., Puzzo, F. n., Pavel-Dinu, M. n., Zhang, F. n., Pillay, S. n., Majzoub, K. n., Tiffany, M. n., Jang, H. n., Sheikali, A. n., Cromer, M. K., Meetei, R. n., Carette, J. E., Porteus, M. H., Pekrun, K. n., Kay, M. A. 2021; 29 (3): 1016–27


    Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by ∼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to ∼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by ∼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by ∼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.

    View details for DOI 10.1016/j.ymthe.2020.10.020

    View details for PubMedID 33678249

  • Improving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards. Nature communications Martin, R. M., Fowler, J. L., Cromer, M. K., Lesch, B. J., Ponce, E., Uchida, N., Nishimura, T., Porteus, M. H., Loh, K. M. 2020; 11 (1): 2713


    Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.

    View details for DOI 10.1038/s41467-020-16455-7

    View details for PubMedID 32483127

  • Proteins Complex of the Fanconi Anemia Pathway as Determinant of AAV-Mediated Genomic Targeted Integration Puzzo, F., de Alencastro, G., Pavel-Dinu, M., Zhang, F., Pillay, S., Majzoub, K., Tiffany, M., Jang, H., Sheikali, A., Cromer, K. M., Meetei, R., Carette, J. E., Porteus, M. H., Pekrun, K., Kay, M. A. CELL PRESS. 2020: 459
  • 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. K., 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


    Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

    View details for PubMedID 31051134

  • Towards The Clinical Translation of Gene Correction in Hematopoietic Stem Cells for Sickle Cell Disease Treatment Lattanzi, A., Dever, D. P., Camarena, J., Lahiri, P., Segal, H., Talbott, N., Srifa, W., Cromer, K., Lee, C., Bao, G., Bathia, N., Uchida, N., Tisdale, J. F., Porteus, M. H. CELL PRESS. 2019: 448
  • Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nature medicine Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Camarena, J., Lemgart, V. T., Cromer, M. K., Vakulskas, C. A., Collingwood, M. A., Zhang, L., Bode, N. M., Behlke, M. A., Dejene, B., Cieniewicz, B., Romano, R., Lesch, B. J., Gomez-Ospina, N., Mantri, S., Pavel-Dinu, M., Weinberg, K. I., Porteus, M. H. 2019


    The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

    View details for PubMedID 30692695

  • 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


    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 β-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 β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

    View details for PubMedID 30195800

  • 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
  • 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


    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

  • Neomorphic effects of recurrent somatic mutations in Yin Yang 1 in insulin-producing adenomas. Proceedings of the National Academy of Sciences of the United States of America Cromer, M. K., Choi, M. n., Nelson-Williams, C. n., Fonseca, A. L., Kunstman, J. W., Korah, R. M., Overton, J. D., Mane, S. n., Kenney, B. n., Malchoff, C. D., Stalberg, P. n., Akerström, G. n., Westin, G. n., Hellman, P. n., Carling, T. n., Björklund, P. n., Lifton, R. P. 2015; 112 (13): 4062–67


    Insulinomas are pancreatic islet tumors that inappropriately secrete insulin, producing hypoglycemia. Exome and targeted sequencing revealed that 14 of 43 insulinomas harbored the identical somatic mutation in the DNA-binding zinc finger of the transcription factor Yin Yang 1 (YY1). Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that this T372R substitution changes the DNA motif bound by YY1. Global analysis of gene expression demonstrated distinct clustering of tumors with and without YY1(T372R) mutations. Genes showing large increases in expression in YY1(T372R) tumors included ADCY1 (an adenylyl cyclase) and CACNA2D2 (a Ca(2+) channel); both are expressed at very low levels in normal β-cells and show mutation-specific YY1 binding sites. Both gene products are involved in key pathways regulating insulin secretion. Expression of these genes in rat INS-1 cells demonstrated markedly increased insulin secretion. These findings indicate that YY1(T372R) mutations are neomorphic, resulting in constitutive activation of cAMP and Ca(2+) signaling pathways involved in insulin secretion.

    View details for DOI 10.1073/pnas.1503696112

    View details for PubMedID 25787250

    View details for PubMedCentralID PMC4386328

  • Identification of somatic mutations in parathyroid tumors using whole-exome sequencing. The Journal of clinical endocrinology and metabolism Cromer, M. K., Starker, L. F., Choi, M. n., Udelsman, R. n., Nelson-Williams, C. n., Lifton, R. P., Carling, T. n. 2012; 97 (9): E1774–81


    The underlying molecular alterations causing sporadic parathyroid adenomas that drive primary hyperparathyroidism have not been thoroughly defined.The aim of the study was to investigate the occurrence of somatic mutations driving tumor formation and progression in sporadic parathyroid adenoma using whole-exome sequencing.Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Selected genes were analyzed for mutations in an additional 185 parathyroid adenomas.Four of eight tumors displayed a frame shift deletion or nonsense mutation in MEN1, which was accompanied by loss of heterozygosity of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic EZH2 mutation that was found by exome sequencing.This study confirms the frequent role of the loss of heterozygosity of chromosome 11 and MEN1 gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, EZH2, in endocrine tumorigenesis.

    View details for DOI 10.1210/jc.2012-1743

    View details for PubMedID 22740705

    View details for PubMedCentralID PMC5393442