Member, Maternal & Child Health Research Institute (MCHRI)
Honors & Awards
Meritorious Travel Grant Award, ASGCT 23rd Annual Meeting, Boston (2020)
Meritorious Travel Grant Award, FOCIS Annual Meeting, San Francisco (2018)
Meritorious Travel Grant Award, ASGCT 20th Annual Meeting, Washington DC (2017)
NSF Graduate Student Research Fellowship, Stanford University (2010-2013)
Boards, Advisory Committees, Professional Organizations
Associate Member, American Society of Gene and Cell Therapy (ASGCT) (2017 - Present)
Associate Member, American Society of Hematology (ASH) (2020 - Present)
Member, Keystone Symposia (2019 - Present)
Member, Federation of Clinical Immunology Societies (FOCIS) (2018 - 2018)
Member, Primary Immune Deficiency Treatment Consortium (PIDTC) (2018 - 2018)
Member, Clinical Immunology Society (2018 - 2018)
Doctor of Philosophy, Stanford University, BIO-PHD (2015)
Bachelor of Science, University of California Los Angeles, Microbio,Immuno,Molec Genetics (2004)
Matthew Porteus, Postdoctoral Faculty Sponsor
- Correction to: Gene Editing Rescues in Vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System. Journal of clinical immunology 2021
Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair.
X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91phox protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1-13 or 2-13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1-13 cDNA did not restore physiologic gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2-13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox expression in exon 2-13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.
View details for DOI 10.1038/s41434-021-00251-z
View details for PubMedID 33712802
Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System.
Journal of clinical immunology
Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.
View details for DOI 10.1007/s10875-021-00989-6
View details for PubMedID 33650026
Enhanced Homology-directed Repair for Highly Efficient Gene Editing in Hematopoietic Stem/Progenitor Cells.
Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.
View details for DOI 10.1182/blood.2020008503
View details for PubMedID 33623984
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
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
Lentivector versus CRISPR/Cas9/AAV6 Gene Editing in X-Linked Severe Combined Immunodeficiency CD34(+) Hematopoietic Cells
CELL PRESS. 2020: 355–56
View details for Web of Science ID 000530089301338
A Genomic Editing-Based Therapeutic Approach for RAG2 Deficiency
CELL PRESS. 2020: 55–56
View details for Web of Science ID 000530089300107
Proteins Complex of the Fanconi Anemia Pathway as Determinant of AAV-Mediated Genomic Targeted Integration
CELL PRESS. 2020: 459
View details for Web of Science ID 000530089302154
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
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
- 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 2019; 24 (5): 821-+
- Gene correction for SCID-X1 in long-term hematopoietic stem cells (vol 10, 1634, 2019) NATURE COMMUNICATIONS 2019; 10
- Gene correction for SCID-X1 in long-term hematopoietic stem cells NATURE COMMUNICATIONS 2019; 10
Gene correction for SCID-X1 in long-term hematopoietic stem cells.
2019; 10 (1): 1634
Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.
View details for PubMedID 30967552
Identification of preexisting adaptive immunity to Cas9 proteins in humans.
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
- Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nature communications 2019; 10 (1): 5624
High-efficiency CRISPR induction of t(9;11) chromosomal translocations and acute leukemias in human blood stem cells.
2019; 3 (19): 2825–35
Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.
View details for DOI 10.1182/bloodadvances.2019000450
View details for PubMedID 31582391
Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.
2019; 10 (1): 2021
The original version of this Article omitted the following from the Acknowledgements: "G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721)."This has now been corrected in both the PDF and HTML versions of the Article.
View details for PubMedID 31028274
CRISPR-Mediated Targeted Insertion of Cybb cDNAs into the Cybb Locus for Correction of X-CGD Patient CD34(+) Cells
CELL PRESS. 2018: 233
View details for Web of Science ID 000435342203060
Genome Editing for IL-10 Deficiency in Purified Hematopoietic Stem Cells
CELL PRESS. 2018: 237–38
View details for Web of Science ID 000435342203069
Genome Editing of Long-Term Human Hematopoietic Stem Cells for X-Linked Severe Combined Immunodeficiency
SPRINGER/PLENUM PUBLISHERS. 2018: 365–66
View details for Web of Science ID 000431311600087
A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.
2018; 24 (8): 1216–24
Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.
View details for PubMedID 30082871
CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells.
The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.
View details for DOI 10.1038/nature20134
View details for PubMedID 27820943
- A crisper look at genome editing: RNA-guided genome modification. Molecular therapy : the journal of the American Society of Gene Therapy 2013; 21 (4): 720-722
- A Crisper Look at Genome Editing: RNA-guided Genome Modification MOLECULAR THERAPY 2013; 21 (4): 719-721
Reprogramming towards pluripotency requires AID-dependent DNA demethylation
2010; 463 (7284): 1042-U57
Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.
View details for DOI 10.1038/nature08752
View details for Web of Science ID 000275108400028
View details for PubMedID 20027182
View details for PubMedCentralID PMC2906123
SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-kappa B-Dependent Gene Expression and Organismal Life Span
2009; 136 (1): 62-74
Members of the sirtuin (SIRT) family of NAD-dependent deacetylases promote longevity in multiple organisms. Deficiency of mammalian SIRT6 leads to shortened life span and an aging-like phenotype in mice, but the underlying molecular mechanisms are unclear. Here we show that SIRT6 functions at chromatin to attenuate NF-kappaB signaling. SIRT6 interacts with the NF-kappaB RELA subunit and deacetylates histone H3 lysine 9 (H3K9) at NF-kappaB target gene promoters. In SIRT6-deficient cells, hyperacetylation of H3K9 at these target promoters is associated with increased RELA promoter occupancy and enhanced NF-kappaB-dependent modulation of gene expression, apoptosis, and cellular senescence. Computational genomics analyses revealed increased activity of NF-kappaB-driven gene expression programs in multiple Sirt6-deficient tissues in vivo. Moreover, haploinsufficiency of RelA rescues the early lethality and degenerative syndrome of Sirt6-deficient mice. We propose that SIRT6 attenuates NF-kappaB signaling via H3K9 deacetylation at chromatin, and hyperactive NF-kappaB signaling may contribute to premature and normal aging.
View details for DOI 10.1016/j.cell.2008.10.052
View details for PubMedID 19135889
SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin
2008; 452 (7186): 492-U16
The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.
View details for DOI 10.1038/nature06736
View details for PubMedID 18337721