- Email: [email protected]
Rapidly Generated Viral-Specific T Lymphocytes for Treatment of Viral Infections in Primary Immunodeficiency
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Site Specific Gene Correction of Defects in CD40 Ligand Using the Crispr/Cas9 Genome Editing Platform Caroline Y. Kuo, M.D.1, Megan D. Hoban, M.S.2, Alok V. Joglekar, Ph.D.3, Donald B. Kohn, M.D.4; 1Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA, 2University of California, Los Angeles, Los Angeles, CA, 3Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, 4 Department of Pediatrics and Department of Microbiology, Immunology, and Molecular GeneticsUniversity of California, Los Angeles, Los Angeles, CA. RATIONALE: Definitive treatment for CD40 Ligand defects are currently only achieved with hematopoietic stem cell transplant. As HSCT carries significant risks, there is a clear need for improved therapeutic modalities. Previous studies using CD40L-/- bone marrow corrected by retroviral-vector transfer of CD40L cDNA in mice resulted in abnormal lymphoproliferation. An alternative approach is targeted gene repair using TALENs (Transcription Activator-Like Effector Nucleases) or CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), which target specific DNA sequences and create double-strand breaks, combined with homologous donor sequences serving as repair templates. METHODS: Six TALENs targeting a patient-specific splice site mutation in intron 3 were unsuccessful in creating gene disruption. This is in contrast to TALENs targeting the 5’UTR that achieve up to 31% allelic disruption in cell lines. Recent work has shown that TALENs are sensitive to DNA methylation states and chromatin structure. CRISPRs do not have the same barriers and several guides were designed to target the patient mutation. After electroporation of K562 cells with CRISPR plasmids, allelic disruption was determined by surveyor endonuclease assay. RESULTS: Patient-specific CRISPRs achieved >50% gene disruption in K562 cells. Co-electroporation with a template donor modified to contain a unique restriction enzyme site demonstrated site-specific gene integration by enzyme digest and gel electrophoresis. Electroporation of XHIM primary cells are underway and re-expression of CD40L will be assessed by flow cytometry. CONCLUSIONS: These results show that site-specific modification of the CD40L gene is achievable and that physiologic expression of the endogenous gene could provide a viable therapy for immune reconstitution in XHIM.
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Rapidly Generated Viral-Specific T Lymphocytes for Treatment of Viral Infections in Primary Immunodeficiency Michael Keller, MD1, Patrick Hanley, PhD2, Catherine Bollard, MD2, Conrad R. Cruz, MD, PhD2, Sarah McCormack, BSc2; 1Allergy/Immunology, Children’s National Medical Center, Washington, DC, 2Children’s National Medical Center. RATIONALE: Adoptive immunotherapy using virus-specific cytotoxic T-lymphocyte (VSTs) products has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT). We hypothesize that VST produced via a rapid protocol will improve clinical outcomes in patients with PIDD following HSCT. METHODS: VSTs were cultured from HSCT donors using peptide pulsed antigen presenting cells and cytokines. VSTs were tested for specificity and non-alloreactivity via IFN-g ELISpot and cytotoxicity assays. Patients were followed for 45 days following infusion for toxicity monitoring and for >6 months for antiviral response. RESULTS: Four patients with PIDD who underwent HSCT were recruited to date, and two have been infused with VSTs at 5x10E6 cells/m2/dose. The first patient was treated for CMV viremia which was refractory to ganciclovir and foscarnet following haploidentical HSCT for SCID. After two infusions of VSTs the CMV load reduced from >1,000,000 to <1000 copies/ml. The second patient was treated for CMV viremia after matched sibling HSCT for MHC-II deficiency, and had viral clearance within 1month post-VST infusion. One patient developed Grade I skin GVHD post
VSTs which resolved within 1 week and was associated with the tapering of immune suppression. CONCLUSIONS: VSTs are safe and effective for control of viral infections in patients with PIDD following HSCT. Studies of the efficacy of VSTs in this setting as well as prior to HSCT for PIDD patients are currently ongoing. Alternaria alternata Fungal Exposure Associated with Increased Fractional Exhaled Nitric Oxide Among Children in New York City Nitzan Soffer, PhD1, Brett J. Green, PhD2, Luis M. Acosta, MD1, Adnan Divjan1, Edward Sobek, PhD3, Merissa McGraw-Boitnotte3, Angela R. Lemons2, Rachel Miller, MD4, Andrew Rundle, DrPH5, Judith Jacobson, DrPH5, Inge Goldstein, DrPH5, Matthew S. Perzanowski, PhD1; 1Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY, 2CDC/NIOSH/ ACIB, Morgantown, WV, 3Assured Bio Labs, Oak Ridge, TN, 4Division of Pulmonary, Allergy and Critical Care Medicine, Columbia University, New York, NY, 5Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY. RATIONALE: Home dampness and fungi have been associated with asthma morbidity; however, most supporting evidence is based on subjective reports or sensitization to fungi and not direct measurements of exposure. Further, allergic responses to inhalant allergens may be augmented by combustion byproduct exposure. We hypothesized that fractional exhaled nitric oxide (FeNO), an airway inflammation biomarker, would be associated with the abundance of some domestic dustborne fungal species and that these relationships would be modified by a marker of combustion exposure, elemental carbon (EC). METHODS: As part of the NYC Neighborhood Asthma and Allergy study, 7-8 year-old children were recruited from neighborhoods with higher (11-19%) and lower (3-9%) asthma prevalence. FeNO was measured from asthmatic (n5125) and non-asthmatic (n5103) children, and bedroom floor dust was collected and analyzed by quantitative polymerase chain reaction for 36 fungi (Environmental Relative Moldiness Index panel). A child’s neighborhood annual airborne EC was estimated using NYC Department of Health data. RESULTS: Quantities of nine fungal species differed significantly across neighborhoods with differing asthma prevalence, including A. alternata (P50.010). A. alternata was positively associated with FeNO (b50.063, p50.033) after adjustment for sex, race/ethnicity, dust mite exposure, seroatopy, environmental tobacco smoke, ambient NO and season. This association was present among the children with higher EC exposure (b5 0.098, p50.004), but not those with lower EC (b5 -0.018, p50.65), Pinteraction50.024. CONCLUSIONS: Among NYC children, A. alternata measured in house dust was associated with FeNO, specifically among children with higher combustion byproduct exposure, suggesting a possible interaction between these two exposures on airway inflammation.
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Abstracts AB17
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
Site Specific Gene Correction of Defects in CD40 Ligand Using the Crispr/Cas9 Genome Editing Platform Caroline Y. Kuo, M.D.1, Megan D. Hoban, M.S.2, Alok V. Joglekar, Ph.D.3, Donald B. Kohn, M.D.4; 1Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA, 2University of California, Los Angeles, Los Angeles, CA, 3Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, 4 Department of Pediatrics and Department of Microbiology, Immunology, and Molecular GeneticsUniversity of California, Los Angeles, Los Angeles, CA. RATIONALE: Definitive treatment for CD40 Ligand defects are currently only achieved with hematopoietic stem cell transplant. As HSCT carries significant risks, there is a clear need for improved therapeutic modalities. Previous studies using CD40L-/- bone marrow corrected by retroviral-vector transfer of CD40L cDNA in mice resulted in abnormal lymphoproliferation. An alternative approach is targeted gene repair using TALENs (Transcription Activator-Like Effector Nucleases) or CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), which target specific DNA sequences and create double-strand breaks, combined with homologous donor sequences serving as repair templates. METHODS: Six TALENs targeting a patient-specific splice site mutation in intron 3 were unsuccessful in creating gene disruption. This is in contrast to TALENs targeting the 5’UTR that achieve up to 31% allelic disruption in cell lines. Recent work has shown that TALENs are sensitive to DNA methylation states and chromatin structure. CRISPRs do not have the same barriers and several guides were designed to target the patient mutation. After electroporation of K562 cells with CRISPR plasmids, allelic disruption was determined by surveyor endonuclease assay. RESULTS: Patient-specific CRISPRs achieved >50% gene disruption in K562 cells. Co-electroporation with a template donor modified to contain a unique restriction enzyme site demonstrated site-specific gene integration by enzyme digest and gel electrophoresis. Electroporation of XHIM primary cells are underway and re-expression of CD40L will be assessed by flow cytometry. CONCLUSIONS: These results show that site-specific modification of the CD40L gene is achievable and that physiologic expression of the endogenous gene could provide a viable therapy for immune reconstitution in XHIM.
53
Rapidly Generated Viral-Specific T Lymphocytes for Treatment of Viral Infections in Primary Immunodeficiency Michael Keller, MD1, Patrick Hanley, PhD2, Catherine Bollard, MD2, Conrad R. Cruz, MD, PhD2, Sarah McCormack, BSc2; 1Allergy/Immunology, Children’s National Medical Center, Washington, DC, 2Children’s National Medical Center. RATIONALE: Adoptive immunotherapy using virus-specific cytotoxic T-lymphocyte (VSTs) products has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT). We hypothesize that VST produced via a rapid protocol will improve clinical outcomes in patients with PIDD following HSCT. METHODS: VSTs were cultured from HSCT donors using peptide pulsed antigen presenting cells and cytokines. VSTs were tested for specificity and non-alloreactivity via IFN-g ELISpot and cytotoxicity assays. Patients were followed for 45 days following infusion for toxicity monitoring and for >6 months for antiviral response. RESULTS: Four patients with PIDD who underwent HSCT were recruited to date, and two have been infused with VSTs at 5x10E6 cells/m2/dose. The first patient was treated for CMV viremia which was refractory to ganciclovir and foscarnet following haploidentical HSCT for SCID. After two infusions of VSTs the CMV load reduced from >1,000,000 to <1000 copies/ml. The second patient was treated for CMV viremia after matched sibling HSCT for MHC-II deficiency, and had viral clearance within 1month post-VST infusion. One patient developed Grade I skin GVHD post
VSTs which resolved within 1 week and was associated with the tapering of immune suppression. CONCLUSIONS: VSTs are safe and effective for control of viral infections in patients with PIDD following HSCT. Studies of the efficacy of VSTs in this setting as well as prior to HSCT for PIDD patients are currently ongoing. Alternaria alternata Fungal Exposure Associated with Increased Fractional Exhaled Nitric Oxide Among Children in New York City Nitzan Soffer, PhD1, Brett J. Green, PhD2, Luis M. Acosta, MD1, Adnan Divjan1, Edward Sobek, PhD3, Merissa McGraw-Boitnotte3, Angela R. Lemons2, Rachel Miller, MD4, Andrew Rundle, DrPH5, Judith Jacobson, DrPH5, Inge Goldstein, DrPH5, Matthew S. Perzanowski, PhD1; 1Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY, 2CDC/NIOSH/ ACIB, Morgantown, WV, 3Assured Bio Labs, Oak Ridge, TN, 4Division of Pulmonary, Allergy and Critical Care Medicine, Columbia University, New York, NY, 5Department of Epidemiology, Mailman School of Public Health, Columbia University, New York, NY. RATIONALE: Home dampness and fungi have been associated with asthma morbidity; however, most supporting evidence is based on subjective reports or sensitization to fungi and not direct measurements of exposure. Further, allergic responses to inhalant allergens may be augmented by combustion byproduct exposure. We hypothesized that fractional exhaled nitric oxide (FeNO), an airway inflammation biomarker, would be associated with the abundance of some domestic dustborne fungal species and that these relationships would be modified by a marker of combustion exposure, elemental carbon (EC). METHODS: As part of the NYC Neighborhood Asthma and Allergy study, 7-8 year-old children were recruited from neighborhoods with higher (11-19%) and lower (3-9%) asthma prevalence. FeNO was measured from asthmatic (n5125) and non-asthmatic (n5103) children, and bedroom floor dust was collected and analyzed by quantitative polymerase chain reaction for 36 fungi (Environmental Relative Moldiness Index panel). A child’s neighborhood annual airborne EC was estimated using NYC Department of Health data. RESULTS: Quantities of nine fungal species differed significantly across neighborhoods with differing asthma prevalence, including A. alternata (P50.010). A. alternata was positively associated with FeNO (b50.063, p50.033) after adjustment for sex, race/ethnicity, dust mite exposure, seroatopy, environmental tobacco smoke, ambient NO and season. This association was present among the children with higher EC exposure (b5 0.098, p50.004), but not those with lower EC (b5 -0.018, p50.65), Pinteraction50.024. CONCLUSIONS: Among NYC children, A. alternata measured in house dust was associated with FeNO, specifically among children with higher combustion byproduct exposure, suggesting a possible interaction between these two exposures on airway inflammation.
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Abstracts AB17
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2