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Dive into the research topics where Karen Sommer is active.

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Featured researches published by Karen Sommer.


Science Translational Medicine | 2015

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

Blythe D. Sather; Guillermo S. Romano Ibarra; Karen Sommer; Gabrielle Curinga; Malika Hale; Iram Khan; Swati Singh; Yumei Song; Kamila Gwiazda; Jaya Sahni; Jordan Jarjour; Alexander Astrakhan; Thor A. Wagner; Andrew M. Scharenberg; David J. Rawlings

Therapeutic coding sequences can be targeted to the CCR5 locus of primary human T cells with high efficiency by using megaTAL nuclease and an AAV donor template. Delete and replace Newer gene-editing methods hold promise for correcting human disease but so far have been hampered by low efficiencies when used in primary cells. To address this issue, Sather et al. have devised a more effective way to both disrupt and replace the CCR5 locus in human T cells, a procedure that has already been shown to improve HIV clearance. Serotype 6 of an adeno-associated viral vector worked particularly well for delivery of megaTAL nucleases and homologous donor templates to primary human T cells, achieving efficient gene-editing rates and little toxicity. The megaTALs generate homology-directed repair (rather than previous efforts, which induce nonhomologous end-joining repair) and so was used for both deletion and accurate replacement of the CCR5 locus. The authors demonstrate that chimeric antigen receptors and an HIV fusion inhibitor inserted into the CCR5 locus ameliorate HIV infection in mice and show that their approach also works in CD34+ hematopoietic precursor cells. Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4+ T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)–mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP–modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34+ cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.


Molecular therapy. Methods & clinical development | 2017

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Malika Hale; Baeckseung Lee; Yuchi Honaker; Wai-Hang Leung; Alexandra Grier; Holly M. Jacobs; Karen Sommer; Jaya Sahni; Shaun W. Jackson; Andrew M. Scharenberg; Alexander Astrakhan; David J. Rawlings

Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.


Molecular and Cellular Biology | 2013

Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation

Miguel E. Moreno-García; Karen Sommer; Héctor Rincón-Arano; Michelle Brault; Jun Ninomiya-Tsuji; Lydia Matesic; David J. Rawlings

ABSTRACT Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.


Molecular therapy. Nucleic acids | 2016

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

Guillermo S. Romano Ibarra; Biswajit Paul; Blythe D. Sather; Patrick Younan; Karen Sommer; John P. Kowalski; Malika Hale; Barry L. Stoddard; Jordan Jarjour; Alexander Astrakhan; Hans-Peter Kiem; David J. Rawlings

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patients immune system and provide protection from HIV infection.


Molecular therapy. Methods & clinical development | 2017

Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector

Swati Singh; Iram Khan; Socheath Khim; Brenda Seymour; Karen Sommer; Matthew Wielgosz; Zachary K. Norgaard; Hans-Peter Kiem; Jennifer E. Adair; Denny Liggitt; Arthur W. Nienhuis; David J. Rawlings

Wiskott-Aldrich syndrome (WAS) is a life-threatening immunodeficiency caused by mutations within the WAS gene. Viral gene therapy to restore WAS protein (WASp) expression in hematopoietic cells of patients with WAS has the potential to improve outcomes relative to the current standard of care, allogeneic bone marrow transplantation. However, the development of viral vectors that are both safe and effective has been problematic. While use of viral transcriptional promoters may increase the risk of insertional mutagenesis, cellular promoters may not achieve WASp expression levels necessary for optimal therapeutic effect. Here we evaluate a self-inactivating (SIN) lentiviral vector combining a chromatin insulator upstream of a viral MND (MPSV LTR, NCR deleted, dl587 PBS) promoter driving WASp expression. Used as a gene therapeutic in Was−/− mice, this vector resulted in stable WASp+ cells in all hematopoietic lineages and rescue of T and B cell defects with a low number of viral integrations per cell, without evidence of insertional mutagenesis in serial bone marrow transplants. In a gene transfer experiment in non-human primates, the insulated MND promoter (driving GFP expression) demonstrated long-term polyclonal engraftment of GFP+ cells. These observations demonstrate that the insulated MND promoter safely and efficiently reconstitutes clinically effective WASp expression and should be considered for future WAS therapy.


Molecular Therapy | 2016

316. Successful Editing of the CD40LG Locus in Human Hematopoietic Stem Cells

Gabrielle Curinga; Sarah Leach; Swati Singh; Nick Hubbard; David Hagin; Karen Sommer; Iram Khan; Yumei Song; Troy R. Torgerson; Andy Scharenberg; David J. Rawlings

X-Linked Hyper-IgM Syndrome (X-HIGM) is a genetic disorder caused by mutations in CD40LG that result in the loss of functional CD40L protein on the T cell surface. CD40L is required for T cells to provide “help” to B cells during an immune challenge; thus X-HIGM patients have impaired immunoglobulin class-switching and somatic hypermutation, and suffer recurrent infections. Murine gene transfer and X-HIGM patient studies suggest that safe and effective gene therapies for this disorder need to replicate cell surface expression patterns of WT endogenous CD40L, as well as knock-out expression of the mutant CD40L; requirements that are unlikely to be achievable using viral gene replacement. We recently reported a gene editing approach combining mRNA-delivered TALEN (targeting just upstream of the coding sequence of CD40LG) with an rAAV donor template for homology directed repair, targeting a promoter-less CD40L cDNA to the ATG start codon of the endogenous allele. This approach restored regulated cell surface expression of CD40L to X-HIGM T cells, disrupted expression of the mutant protein, and resulted in T cells that induced B cell class-switching in vitro, thus demonstrating its potential as a T cell therapy for X-HIGM.Although the pathologies of X-HIGM are attributed mostly to the T cell defects, CD40L is expressed in most hematopoietic lineages. Besides providing a stable X-HIGM treatment, editing of autologous hematopoietic stem cells (HSC) would likely rescue regulated CD40L expression in all lineages. A selective advantage for gene corrected cells is not anticipated; however, patients with mixed donor chimerism post-transplantation have substantial improvements when as few as 10% of HSC have the WT allele. Here we report gene editing of CD40LG in adult human CD34+ PBSC at rates that are anticipated to provide such clinical benefit. Initial experiments using mRNA delivery of TALEN pairs targeting exon 1 of the CD40LG locus in human CD34+ cells demonstrated indel frequencies of >50%. To investigate the potential for HDR at the CD40LG locus in adult CD34+ cells, we combined delivery of the TALEN with an AAV6 donor template containing an MND promoter-GFP expression cassette flanked by 1 kb CD40LG homology arms. This donor template allowed us to track editing rates within the CD40LG locus (normally silent in HSCs) by flow cytometry. Using this co-delivery strategy, we consistently achieved editing rates of ~30% across multiple control human stem cell donors. Edited cells demonstrated minimal loss of viability, and expansion rates in culture equivalent to controls. Edited cells have been transplanted into immune deficient, NSG mice in order to track engraftment and differentiation potential. In parallel, we are currently evaluating editing rates using a more clinically relevant, promoter-less donor template encoding the CD40L cDNA. In summary, we have achieved clinically relevant rates of gene editing within the endogenous CD40LG locus in human HSC, setting the stage for additional work required to translate this approach into clinical application.


Molecular Therapy | 2016

761. Targeted Killing of HIV Infected Cells Using CCR5-Disrupted Anti-HIV-CAR T Cells

Guillermo S. Romano Ibarra; Malika Hale; Karen Sommer; Iram Khan; Jaya Sahni; Christopher W. Peterson; Hans-Peter Kiem; Jordan Jarjour; Alexander Astrakhan; Andrew M. Scharenberg; David J. Rawlings; Thor A. Wagner

A cure for HIV remains an important treatment goal for 30 million HIV-infected individuals worldwide. Long term control of HIV following a single treatment will require a mechanism to eradicate the latent reservoir of HIV infected cells that are capable of reactivating. A previous phase II clinical trial using an anti-HIV chimeric antigen receptor (αHIV-CAR) targeting the CD4-binding site on HIV envelope was partially effective. To optimize this approach we have developed a series of CARs based on the scFV of broadly neutralizing HIV antibodies targeting four different structural regions of the HIV envelope: the V1/V2 loop, the V3 loop, the CD4 binding site, and the membrane-proximal external region. αHIV-CAR T cells targeting different epitopes were compared and were able to kill > 80% of HIV-infected cells grown in the presence of ART. However, a limitation of αHIV-CAR T cells is that the αHIV-CAR also serves as a receptor for HIV and allows HIV infection of αHIV-CAR T cells. Therefore we disrupted the major co-receptor required for HIV cell entry, CCR5, using a megaTAL nuclease, as a means of protecting the CAR-expressing cells from HIV infection. We used two strategies for achieving these dual modifications in primary human T cells. For both strategies, the CCR5 megaTAL nuclease was delivered by mRNA electroporation, which has previously been shown to induce a high rate of bi-allelic NHEJ-mediated gene disruption. αHIV-CAR expression cassettes were delivered into the host genome via lentiviral vectors (LV), or were targeted to the megaTAL nuclease cleavage site in CCR5 using an adeno-associated virus (AAV) that included CCR5 homology arms. Both strategies resulted in stable expression of the CAR construct and specific activation of CAR+ T cells in the presence of an HIV+ cell line. In the presence of actively replicating virus, CCR5-megaTAL treated CAR+ T cells out-performed CAR+ T cells generated by LV delivery alone as measured by reduction in HIV capsid protein. To enable testing in non-human primates (NHP), we re-optimized the cell-editing protocol with NHP lymphocytes. Primary T cells from pigtail macaques were successfully transfected with CCR5 megaTAL mRNA and achieved a CCR5 disruption rate of 50%. Cells were subsequently transduced with αHIV-CAR LV resulting in ~70% of manipulated cells with αHIV-CAR. Expansion of the cells in vitro resulted in 60-fold expansion over 8 days. CAR+ NHP cells specifically killed HIV infected human cells demonstrating that NHP-derived αHIV-CAR+ T cells retained killing function. In conclusion, it is feasible to construct αHIV-CAR+ T cells that are protected from HIV infection in human and NHP cells, and warrants further study in vivo.


Molecular Therapy | 2015

482. Editing of the CD40L Gene Restores Regulated CD40L Expression in X-HIGM Patient T Cells

Nick Hubbard; Yumei Song; David Hagin; Karen Sommer; Andrew M. Scharenberg; Troy Torgerson; David J. Rawlings

X-linked hyper-IgM syndrome (X-HIGM) is a primary immunodeficiency syndrome characterized by insufficient CD40 Ligand (CD40L) expression, resulting in defective T-cell help, impaired immunoglobulin class-switching and recurrent opportunistic infections. Previously, constitutive CD40L expression in murine bone marrow cells and thymocytes was achieved by retroviral gene transfer and successfully corrected immune function. However, treated animals developed severe lymphoproliferative disease, likely due to constitutive CD40L surface expression. This demonstrated the importance of preserving endogenous gene regulation in this and other gene therapy endeavors. Here, we report efficient, on-target Homology Directed Repair (HDR) editing of the CD40L locus in primary human T cells using a combination of TALEN gene targeting and a donor template delivered transiently by recombinant Adeno-Associated Virus (rAAV). Our TALEN and donor template reagents were designed to insert a coding sequence (GFP or CD40L) upstream of the endogenous translation start site within Exon 1, thus allowing transgene expression to be regulated by the endogenous CD40L promoter, and included the CD40L 3’UTR to preserve known post-transcriptional regulation features. The kinetics of GFP and edited CD40L surface expression after PMA-Ionomycin stimulation paralleled that of endogenous CD40L in unedited T-cells. Additionally, activated T-cells expressing edited CD40L were able to bind CD40-Ig similar to unedited cells. When T-cells from X-HIGM patients were edited in this fashion, CD40L expression and CD40-Ig binding was restored, and repaired activated X-HIGM T-cells induced the expression of early activation markers and class switching of naive B-cells in vitro. Finally, we found no differences in TCR repertoire or engraftment and stability in NSG mice between T cells that underwent gene editing vs. unedited cells. These results demonstrate the feasibility of site-directed gene repair to restore normally regulated CD40L protein expression and functional T-cell help.


Molecular Therapy | 2015

238. BTK-Promoter LV Vectors Utilizing Conserved Intron Element Mediate Functional Rescue in Murine XLA

Swati Singh; Brenda Seymour; Karen Sommer; Iram Khan; Socheath Khim; Malika Hale; Courtnee Clough; David J. Rawlings

X-linked agammaglobulinema (XLA) is a rare immunodeficiency disorder caused by a mutation in the gene coding Brutons tryosine kinase (BTK). BTK is required for signal transduction downstream of the B cell antigen receptor (BCR) and is essential for normal B cell development, activation, and survival. Patients lacking BTK have very few mature B cells, markedly reduced antibody production, and suffer from recurrent and life-threatening infections. XLA is an ideal candidate for hematopoietic gene therapy, as B cells that express BTK are positively selected during development. We previously developed a SIN-lentiviral (LV) construct that used the human BTK promoter (BTKp) to drive BTK expression. We also incorporated a truncated, 0.7 kb, fragment derived from the HNRPA2B1/CBX3 ubiquitously acting chromatin opening element (UCOE) upstream of the BTKp to facilitate stable BTK expression over time. Bone marrow transplantation in a murine XLA model (BTK-/-Tec-/- mice) using LV transduced hematopoietic stem cells (HSCs) reconstituted myeloid and B cell numbers, BCR-dependent B cell proliferation and T-dependent antibody responses. The goal of the current study was to further optimize BTK expression in B and myeloid cells while reducing the viral copy number (VCN) required for efficient functional reconstitution. Using the ENCODE database, we identified several highly conserved upstream and intronic regions exhibiting histone modifications and transcription factor binding profiles in B cells consistent with enhancer regions. These conserved elements were introduced individually or in tandem into the LV construct between the UCOE and BTKp and tested for their ability to rescue BTK expression and B cell development in the murine XLA model. Addition of two key intronic regions led to consistent BTK expression and rescued the development and function of B cells, despite a >5-fold reduction in VCN per splenic B cell. Our findings illustrate the usefulness of this strategy for improving the efficacy of endogenous promoter-based genetic therapies. This new LV vector, UCOE. I4-5.BTKpro-hBTK, is currently being evaluated using mobilized CD34+ stem cells isolated from XLA patients and in additional pretoxicology studies designed to support its application in a future LV clinical trial for XLA.


Molecular Therapy | 2015

685. Efficient Targeted Gene Modification in Primary Human Hematopoietic Cells Using Co-Delivery of Nuclease mRNA and AAV Donors

Guillermo S. Romano Ibarra; Gabrielle Curinga; Blythe D. Sather; Karen Sommer; Malika Hale; Iram Khan; Swati Singh; Youmei Song; Kamila Gwiazda; Jaya Sahni; Socheath Khim; Alexander Astrakhan; Thor A. Wagner; Andrew M. Scharenberg; David J. Rawlings

Current clinical HIV treatment strategies applying gene-editing technologies to disrupt the HIV co-receptor CCR5 via nuclease delivery are limited by inefficient bi-allelic disruption and the lack of a combinatorial approach to control HIV infection. The ability to target additional factors conferring HIV resistance to the CCR5 locus with efficient bi-allelic disruption has the potential to translate to improved clinical outcomes. Here we demonstrate a novel method in which RNA-based nuclease expression is paired with AAV-mediated delivery of a homologous gene-targeting donor template to achieve highly efficient targeted recombination in primary human T-cells. This method consistently achieves an average of 60% rates of homology directed recombination into the CCR5 locus in T-cells (>20 experiments with multiple donors); with up to 90% of the gene-modified population exhibiting bi-allelic modification. T-cells modified with this protocol maintain a diverse repertoire and engraft in immune deficient, NOD/SCID/gc-/- (NSG) mice as efficiently as unmodified control T-cells. Using this method, we have integrated chimeric antigen receptors (CARs) into the CCR5 locus; and show that the resulting “targeted CAR” or tCAR T-cells exhibit anti-tumor activities indistinguishable from those generated using lentiviral random integration. Alternatively, we introduced the C46 HIV fusion inhibitor by targeted recombination generating T cell populations with very high-rates of bi-allelic CCR5 disruption paired with protection from HIV with CXCR4 co-receptor tropism. Next, as proof of principle, we utilized the CCR5 site as a ‘safe harbor’ locus for targeted gene addition of the human Wiskott-Aldrich syndrome (WAS) gene. Finally, this novel protocol was also successfully applied to adult human mobilized CD34+ cells, resulting in 10-20% homologous gene targeting, the first such report in adult human CD34+ cells at the CCR5 locus. Our results demonstrate that high-efficiency targeted integration is feasible in primary human cells, and highlight the potential of gene editing technologies to engineer CAR T-cells or other T cell products with myriad novel functional properties.

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Dive into the Karen Sommer's collaboration.

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Andrew M. Scharenberg

Seattle Children's Research Institute

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Iram Khan

Seattle Children's Research Institute

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Malika Hale

Seattle Children's Research Institute

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Guillermo S. Romano Ibarra

Seattle Children's Research Institute

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Jaya Sahni

Seattle Children's Research Institute

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Swati Singh

Seattle Children's Research Institute

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Jordan Jarjour

Fred Hutchinson Cancer Research Center

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Socheath Khim

Seattle Children's Research Institute

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