First-in-human in vivo genome editing via AAV-zinc-finger nucleases for mucopolysaccharidosis I/II and hemophilia B.

Zinc-finger nuclease (ZFN)-based in vivo genome editing is a novel treatment that can potentially provide lifelong protein replacement with single intravenous administration. Three first-in-human open-label ascending single-dose phase 1/2 studies were performed in parallel (starting November 2017) primarily to assess safety and tolerability of ZFN in vivo editing therapy in mucopolysaccharidosis I (MPS I) (n = 3), MPS II (n = 9), and hemophilia B (n = 1). Treatment was well tolerated with no serious treatment-related adverse events. At the 1e13 vg/kg dose, evidence of genome editing was detected through albumin-transgene fusion transcripts in liver for MPS II (n = 2) and MPS I (n = 1) subjects. The MPS I subject also had a transient increase in leukocyte iduronidase activity to the lower normal range. At the 5e13 vg/kg dose, one MPS II subject had a transient increase in plasma iduronate-2-sulfatase approaching normal levels and one MPS I subject approached mid-normal levels of leukocyte iduronidase activity with no evidence of genome editing. The hemophilia B subject was not able to decrease use of factor IX concentrate; genome editing could not be assessed. Overall, ZFN in vivo editing therapy had a favorable safety profile with evidence of targeted genome editing in liver, but no long-term enzyme expression in blood.

Correction: Blood transcriptional profiling reveals IL-1 and integrin signaling pathways associated with clinical response to extracorporeal photopheresis in patients with leukemic cutaneous T-cell lymphoma.

[This corrects the article DOI: 10.18632/oncotarget.26900.].

Blood transcriptional profiling reveals IL-1 and integrin signaling pathways associated with clinical response to extracorporeal photopheresis in patients with leukemic cutaneous T-cell lymphoma.

Extracorporeal photopheresis (ECP) is a frontline therapy for patients with leukemic cutaneous T-cell lymphoma (L-CTCL), but its mechanisms of action are not fully understood. This study was to explore the molecular mechanisms underlying clinical response versus non-response in patients with L-CTCL. We performed blood transcriptional profiling of ten L-CTCL patients at Day 2 and 1 month post- ECP compared to pre-ECP baseline using Agilent Whole Human Genome Microarray technology. Differentially expressed genes (DEGs) between five clinically-responsive patients and five clinically-resistant patients were cross-compared. Higher numbers of genes were modulated in responders than non-responders after ECP at both Day 2 and 1 month, with two thirds of DEGs down-regulated. The down-regulated DEGs at 1 month post-ECP were related to inflammatory, immune and/or stress responses, platelet functions, and chromatin remodeling. Upregulated DEGs were mainly related to functions of the nucleolus. Pathway analysis revealed that integrin and IL-1 signaling pathways were the top pathways affected in responders, which were minimally affected in non-responders. The top upstream transcription regulators affected were IL1B, EGR1, FAS, and TGFB1. Our results suggest that the modulation of cell adhesion and suppression of IL-1ß induced inflammation may underlie the efficacy of ECP in L-CTCL.

The effect of extracorporeal photopheresis alone or in combination therapy on circulating CD4(+) Foxp3(+) CD25(-) T cells in patients with leukemic cutaneous T-cell lymphoma.

PURPOSE: Extracorporeal photopheresis (ECP) alone or in combination therapy is effective for treatment of leukemic cutaneous T-cell lymphoma (L-CTCL), but its mechanism(s) of action remain unclear. This study was designed to investigate the effect of ECP on regulatory T cells and CD8(+) T cells in L-CTCL patients. EXPERIMENTAL DESIGN: Peripheral blood from 18 L-CTCL patients at baseline, Day 2, 1 month, 3 month, and 6 month post-ECP therapy was analyzed by flow cytometry for CD4(+) CD25(+/high) , CD4(+) Foxp3(+) CD25(+/-) , CD3(+) CD8(+) , CD3(+) CD8(+) CD69(+) , and CD3(+) CD8(+) IFN-γ(+) T cells. Clinical responses were assessed and correlated with changes in these T-cell subsets. RESULTS: Twelve of 18 patients achieved clinical responses. The average baseline number of CD4(+) CD25(+/high) T cells of PBMCs in L-CTCL patients was normal (2.2%), but increased at 6-month post-therapy (4.3%, P < 0.01). The average baseline number of CD4(+) Foxp3(+) T cells out of CD4(+) T cells in nine evaluable patients was high (66.8 ± 13.7%), mostly CD25 negative. The levels of CD4(+) Foxp3(+) T cells in responders were higher (n = 6, 93.1 ± 5.7%) than nonresponders (n = 3, 14.2 ± 16.0%, P < 0.01), and they declined in parallel with malignant T cells. The numbers of CD3(+) CD8(+) CD69(+) and CD3(+) CD8(+) IFN-γ(+) T cells increased at 3-month post-therapy in five of six patients studied. CONCLUSIONS: Extracorporeal photopheresis alone or in combination therapy might be effective in L-CTCL patients whose malignant T cells have a CD4(+) Foxp3(+) CD25(-) phenotype.

Extracorporeal photopheresis in the treatment of graft-versus-host disease: evidence and opinion.

Despite significant advances in prevention and treatment strategies, graft-versus-host disease remains the most significant cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cellular transplantation. Corticosteroids remain the standard frontline therapy for graft-versus-host disease; however, a considerable number of patients will not respond adequately and others will be significantly affected by adverse effects. Extracorporeal photopheresis is one of several secondary therapies which have shown promise in the clinical setting. While the procedure itself has been around for over 20 years, our understanding of the mechanisms from which therapeutic benefits are seen, and the population they are seen in, remains limited. In this article, we review the use of extracorporeal photopheresis for the treatment of graft-versus-host disease including details covering the procedure's mechanism of action, safety profile and clinical efficacy data.

Sézary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-beta on the cell surface.

Because syndecan-4 (SD-4) on effector and memory T cells inhibits T-cell activation by binding dendritic cell-associated heparan sulfate proteoglycan-integrin ligand (DC-HIL) on antigen presenting cells and because malignant cells of the cutaneous T-cell lymphoma (CTCL) subset, Sézary syndrome (SS), exhibit memory T-cell phenotype, we posited SS cells to express SD-4. Indeed, malignant T cells from patients with SS and from CTCL cell lines constitutively expressed SD-4 at high levels, in contrast to T cells from healthy volunteers and patients with other inflammatory skin diseases and to non-CTCL cell lines that did not. SS cells also bound to DC-HIL at a level higher than normal T cells activated in vitro, resulting in their inhibited proliferation to anti-CD3 antibody. SD-4 on SS cells also trapped transforming growth factor-ß1 to their cell surface, enhancing their ability to inhibit activation of syngeneic and allogeneic normal T cells. All of these inhibitory properties were dependent on overexpression of distinct heparan sulfate (HS) moieties by SD-4 on SS cells. Finally, we showed toxin-conjugated DC-HIL to abrogate the ability of SS cells to proliferate in vitro. These findings indicate that SD-4 bearing distinct HS moieties plays a pathogenic role in SS and may be targeted for treatment.

Induction of T-cell responses against cutaneous T-cell lymphomas ex vivo by autologous dendritic cells transfected with amplified tumor mRNA.

Sézary syndrome (SzS), the leukemic variant of cutaneous T-cell lymphomas, is incurable. Dendritic cells (DCs) transfected with tumor mRNA can stimulate antitumor immunity in certain cancer patients. In this study, we determined whether mRNAs from Sézary cells could be used for loading DCs and stimulating antitumor immunity. Autologous DCs were generated from monocytes of the peripheral blood from 10 patients with SzS. Total RNA was extracted from Sézary cells and amplified by T7 in vitro transcription. The induction of antitumor IFN-gamma and granzyme B (GrB)-producing cytotoxic T lymphocytes (CTL) by RNA-transfected DCs was determined by ELISPOT assays. We found that IFN-gamma was required for IL-12p70 production by monocyte-derived DCs from SzS. The oncogenic transcription factor Twist and the tyrosine kinase receptor EphA4 were expressed in total RNA from Sézary cells and the paired amplified mRNAs. RNA-transfected DCs induced antitumor IFN-gamma-producing CTLs in 7 of 10 subjects and GrB-producing CTLs in 6 of 9 subjects. Both CD3+CD8+ T cells and CD4+CD25+ T cells were expanded without induction of regulatory T cells. These data support the concept of using tumor mRNA for a vaccine strategy that requires small amounts of tumor cells without need for specific antigens in patients with SzS.