The British Society for Gene and Cell Therapy at 20 (2003-2023)

2023 marks the 20th anniversary of the British Society for Gene and Cell Therapy. In these 20 years, the field of gene and cell therapy has gone from promising strategy to clinical reality. This report describes the history, objectives, organisation and activities of BSGCT to advance research and practice of gene and cell therapy in the UK

An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cell populations

Gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat hematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and their in vitro expansion for clinical application. In this study, we assessed the effect of different culture medium compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex vivo culture. We demonstrated that by supplementing the culture medium with stem cell agonists and by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion. Overall, the implementation of this optimized ex vivo HSPC culture protocol can improve the efficacy, feasibility and applicability of gene editing as a key step to unlocking the full therapeutic potential of this powerful technology

Polyphenon E enhances the antitumor immune response in neuroblastoma by inactivating myeloid suppressor cells

This is the author's accepted manuscript. The final published article is available from the link below. Note: In this manuscript as well as in the original published version of this article the word "Polyphenon" was incorrectly spelled in the title as "Polyphenol."Purpose: Neuroblastoma is a rare childhood cancer whose high risk, metastatic form has a dismal outcome in spite of aggressive therapeutic interventions. The toxicity of drug treatments is a major problem in this pediatric setting. In this study, we investigated whether Polyphenon E, a clinical grade mixture of green tea catechins under evaluation in multiple clinical cancer trials run by the National Cancer Institute (Bethesda, MD), has anticancer activity in mouse models of neuroblastoma. Experimental Design: We used three neuroblastoma models: (i) transgenic TH-MYCN mouse developing spontaneous neuroblastomas; (ii) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenotransplanted with human SHSY5Y cells; and (iii) A/J mice transplanted with syngeneic Neuro 2A cells. Mice were randomized in control and Polyphenon E–drinking groups. Blood from patients with neuroblastoma and normal controls was used to assess the phenotype and function of myeloid cells. Results: Polyphenon E reduced the number of tumor-infiltrating myeloid cells, and inhibited the development of spontaneous neuroblastomas in TH-MYCN transgenic mice. In therapeutic models of neuroblastoma in A/J, but not in immunodeficient NOD/SCID mice, Polyphenon E inhibited tumor growth by acting on myeloid-derived suppressor cells (MDSC) and CD8 T cells. In vitro, Polyphenon E impaired the development and motility of MDSCs and promoted differentiation to more neutrophilic forms through the 67 kDa laminin receptor signaling and induction of granulocyte colony-stimulating factor. The proliferation of T cells infiltrating a patient metastasis was reactivated by Polyphenon E. Conclusions: These findings suggest that the neuroblastoma-promoting activity of MDSCs can be manipulated pharmacologically in vivo and that green tea catechins operate, at least in part, through this mechanism.SPARKS, Research in Childhood Cancer, the CGD Research Trust, and the Wellcome Trust

Integrase-deficient lentiviral vectors mediate efficient gene transfer to human vascular smooth muscle cells with minimal genotoxic risk

We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer

Human involucrin promoter mediates repression-resistant and compartment-specific LEKTI expression

Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector–mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral–derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter–transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type–related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice.We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization

Critical role of WASp in germinal center tolerance through regulation of B cell apoptosis and diversification

A main feature of Wiskott-Aldrich syndrome (WAS) is increased susceptibility to autoimmunity. A key contribution of B cells to development of these complications has been demonstrated through studies of samples from affected individuals and mouse models of the disease, but the role of the WAS protein (WASp) in controlling peripheral tolerance has not been specifically explored. Here we show that B cell responses remain T cell dependent in constitutive WASp-deficient mice, whereas selective WASp deletion in germinal center B cells (GCBs) is sufficient to induce broad development of self-reactive antibodies and kidney pathology, pointing to loss of germinal center tolerance as a primary cause leading to autoimmunity. Mechanistically, we show that WASp is upregulated in GCBs and regulates apoptosis and plasma cell differentiation in the germinal center and that the somatic hypermutation-derived diversification is the basis of autoantibody development

Critical role of WASp in germinal center tolerance through regulation of B cell apoptosis and diversification

This project was supported by grant 310030-179251 from the Suisse National Science Foundation (SNF) (to F.C.), funds from the BLACKSWAN Foundation (BSF-005) (to M.D.), and the Wellcome Trust (to A.J.T.).A main feature of Wiskott-Aldrich syndrome (WAS) is increased susceptibility to autoimmunity. A key contribution of B cells to development of these complications has been demonstrated through studies of samples from affected individuals and mouse models of the disease, but the role of the WAS protein (WASp) in controlling peripheral tolerance has not been specifically explored. Here we show that B cell responses remain T cell dependent in constitutive WASp-deficient mice, whereas selective WASp deletion in germinal center B cells (GCBs) is sufficient to induce broad development of self-reactive antibodies and kidney pathology, pointing to loss of germinal center tolerance as a primary cause leading to autoimmunity. Mechanistically, we show that WASp is upregulated in GCBs and regulates apoptosis and plasma cell differentiation in the germinal center and that the somatic hypermutation-derived diversification is the basis of autoantibody development.Publisher PDFPeer reviewe

Lentiviral Gene Transfer Corrects Immune Abnormalities in XIAP Deficiency

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) deficiency is a severe immunodeficiency with clinical features including hemophagocytic lymphohistiocytosis (HLH) and inflammatory bowel disease (IBD) due to defective NOD2 responses. Management includes immunomodulatory therapies and hematopoietic stem cell transplant (HSCT). However, this cohort is particularly susceptible to the chemotherapeutic regimens and acutely affected by graft-vs-host disease (GvHD), driving poor long-term survival in transplanted patients. Autologous HSC gene therapy could offer an alternative treatment option and would abrogate the risks of alloreactivity. METHODS: Hematopoietic progenitor (Lin-ve) cells from XIAPy/- mice were transduced with a lentiviral vector encoding human XIAP cDNA before transplantation into irradiated XIAP y/- recipients. After 12 weeks animals were challenged with the dectin-1 ligand curdlan and recovery of innate immune function was evaluated though analysis of inflammatory cytokines, body weight, and splenomegaly. XIAP patient-derived CD14+ monocytes were transduced with the same vector and functional recovery was demonstrated using in vitro L18-MDP/NOD2 assays. RESULTS: In treated XIAPy/- mice, ~40% engraftment of gene-corrected Lin-ve cells led to significant recovery of weight loss, splenomegaly, and inflammatory cytokine responses to curdlan, comparable to wild-type mice. Serum IL-6, IL-10, MCP-1, and TNF were significantly reduced 2-h post-curdlan administration in non-corrected XIAPy/- mice compared to wild-type and gene-corrected animals. Appropriate reduction of inflammatory responses was observed in gene-corrected mice, whereas non-corrected mice developed an inflammatory profile 9 days post-curdlan challenge. In gene-corrected patient CD14+ monocytes, TNF responses were restored following NOD2 activation with L18-MDP. CONCLUSION: Gene correction of HSCs recovers XIAP-dependent immune defects and could offer a treatment option for patients with XIAP deficiency

The cytoplasm of living cells behaves as a poroelastic material

The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at physiologically relevant timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology

Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients