We have assessed the utility of 3-dimensional (3D) in vitro human cell models to understand barriers to chimeric antigen receptor (CAR)-T cell activity in high-grade serous ovarian cancer, (HGSOC) a disease with a poor response to immunotherapy. We defined mucin-1 (MUC1) as a potential target in HGSOC biopsies and the HGSOC cell lines, OvCAR3 and G164. We then generated CAR-T cells against MUC1 and tested them in spheroid and collagen gel cultures. In spheroids, although CAR-T cells killed OvCAR3 cells, G164 cells failed to induce CAR-T cell activation or cytotoxicity. However, when we added primary omental fibroblasts from ovarian cancer patients to G164 spheroids, CAR-T cells were activated and cytotoxic. Fibroblast conditioned medium also activated CAR-T cells to kill G164 cells in spheroids and this was due to their production of C-C motif chemokine ligand 2 (CCL2). Further experiments revealed that CCL2 produced by fibroblast stimulated CCR2/4 positive CAR-T cells to a higher state of activation, which enhanced the cytotoxicity of CAR-T cells against G164 cells. We then investigated CAR-T cell activity in co-cultures of OvCAR3 or G164 cells and primary fibroblasts embedded in collagen. CAR-T cells migrated into OvCAR3 gels and killed the malignant cells during a three-day period. However, CAR-T cells failed to migrate into gels with G164 cells and there was no malignant cell killing. Gels containing G164 cells had denser extracellular matrix (ECM) than OvCAR3 gels, as measured by staining for collagens and fibronectin. Previously, we showed that transforming growth factor-beta (TGFβ) secreted by HGSOC cells acted on fibroblasts to induce the production of ECM in collagen gels.1 Treating G164 gels with the TGFβ receptor inhibitor SB431542 reduced ECM density, stimulated CAR-T cell migration and restored CAR-T cell cytotoxicity against G164 cells. Using these different human 3D models we have demonstrated that malignant cell intrinsic factors can cause resistance to CAR-T cells. Sensitivity to CAR-T cell killing can be modulated both positively and negatively by fibroblasts. Targeting ECM along with CAR-T cell therapy might improve the efficiency of CAR-T cells in solid tumors. 1Delaine-Smith et al, iScience, 2021 Citation Format: Joash Dominic Joy, Beatrice Malacrida, Florian Laforêts, Panoraia Kotantaki, Eleni Maniati, Sarah Hopkins, Ianire Calleja, Sara Brett, Takis Athanasopoulos, Sadfer Ali, Peter Emery-Billcliff, Ida Ricciardelli, Charlotte Kay, Jayne Colebrook, Magda Ali, Katherine Strong, Frances Balkwill. TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 693.

Gene therapy is a relatively novel field that amounts to around four decades of continuous growth with its good and bad moments. Currently, the field has entered the clinical arena with the ambition to fulfil its promises for a permanent fix of incurable genetic disorders. Hemoglobinopathies as target diseases and hematopoietic stem cells (HSCs) as target cells of genetic interventions had a major share in the research effort toward efficiently implementing gene therapy. Dissection of HSC biology and improvements in gene transfer and gene expression technologies evolved in an almost synchronous manner to a point where the two fields seem to be functionally intercalated. In this review, we focus specifically on the development of gene therapy for hemoglobin disorders and look at both gene addition and gene correction strategies that may dominate the field of HSC-directed gene therapy in the near future and transform the therapeutic landscape for genetic diseases.

Cell and gene therapies have achieved impressive results in the treatment of rare genetic diseases using gene corrected stem cells and haematological cancers using chimeric antigen receptor T cells. However, these two fields face significant challenges such as demonstrating long-term efficacy and safety, and achieving cost-effective, scalable manufacturing processes. The use of small molecules is a key approach to overcome these barriers and can benefit cell and gene therapies at multiple stages of their lifecycle. For example, small molecules can be used to optimise viral vector production during manufacturing or used in the clinic to enhance the resistance of T cell therapies to the immunosuppressive tumour microenvironment. Here, we review current uses of small molecules in cell and gene therapy and highlight opportunities for medicinal chemists to further consolidate the success of cell and gene therapies.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

The simian immunodeficiency virus (SIV) macaque model represents the best animal model for testing new human immunodeficiency virus type 1 (HIV-1) vaccines. Previous studies employing replication-defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with an rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to the envelope and neutralize the virus or mediate antibody-dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established has also shown some success. In contrast, the vaccine described here mediates resistance by a different mechanism from that described above, which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.

Advanced Textbook on Gene Transfer, Gene Therapy and Genetic Pharmacology, pp. 503-526 (2019) No Access27: GENETIC THERAPY OF MUSCLE DISEASES: DUCHENNE MUSCULAR DYSTROPHYTakis Athanasopoulos, Susan Jarmin, Helen Foster, Keith Foster, Jagjeet Kang, Taeyoung Koo, Alberto Malerba, Linda Popplewell, Daniel Scherman, and George DicksonTakis AthanasopoulosSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Susan JarminSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Helen FosterSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Keith FosterSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Jagjeet KangSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Taeyoung KooSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Alberto MalerbaSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Linda PopplewellSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK, Daniel SchermanLaboratory of Chemical and Genetic Pharmacology and of Biomedical Imaging, Paris Descartes Pharmacy University, CNRS, Inserm, Chimie ParisTech, 4, avenue de l'Observatoire Paris Cedex 06, France, and George DicksonSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UKhttps://doi.org/10.1142/9781786346889_0027Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: The following sections are included: Introduction Attractiveness of Muscle Tissue for Gene Therapy Approaches Genetic Pharmacology of the Mutated Dystrophin Pre-mRNA by Exon Skipping for Duchenne Muscular Dystrophy Treatment Microdystrophin and Myostatin Gene Therapy for Duchenne Muscular Dystrophy Using Adeno-associated Virus Vectors, Gene Therapy: Administration of the Dystrophin Gene and/or of a Protein Inhibiting Myostatin References FiguresReferencesRelatedDetails Advanced Textbook on Gene Transfer, Gene Therapy and Genetic PharmacologyMetrics History PDF download

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients.

Gene delivery vectors that do not rely on host cell genome integration offer several advantages for gene transfer, chiefly the avoidance of insertional mutagenesis and position effect variegation. However, unless engineered for replication and segregation, nonintegrating vectors will dilute progressively in proliferating cells, and are not exempt of epigenetic effects. This article provides an overview of the main nonintegrating viral (adenoviral, adeno-associated viral, integration-deficient retro-lentiviral, poxviral), and nonviral (plasmid vectors, artificial chromosomes) vectors used for preclinical and clinical cell and gene therapy applications. Particular emphasis is placed on their use in hematologic disease.

Duchenne Muscular Dystrophy (DMD) is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno-Associated Virus vectors (rAAV) hold great promise and allow very efficient transduction of skeletal and cardiac muscles. However, full-length dystrophin cDNA exceeds the packaging capacity for a single rAAV gene-delivery cassette. Therefore, truncated versions namely micro-dystrophins have been designed and optimized to contain few clinically important regions of the dystrophin protein. We have tested a rAAV2/8 vector encoding a sequence optimised canine micro-dystrophin transgene, driven by a muscle-synthetic Spc512 promoter (rAAV2/8-Spc512-µDys) in a total of 12 Golden Retriever Muscular Dystrophy (GRMD) dogs, the canine model of DMD. Isolated limb perfusion studies using a single administration of vector induced high levels of micro-dystrophin expression in the treated limb (up to 90% dystrophin positive fibres) with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. Similarly, single-dose intravascular delivery of the same rAAV2/8-Spc512-µDys, in absence of immunosuppression, led to long-term transduction of distant muscle groups and extended lifespan (up to 2 years). Profound improvement of multiple clinical features was observed, including gait and respiratory parameters and no toxicity or deleterious humoral and/or cell-mediated immune responses were observed. This study demonstrates the safety and long term efficacy of rAAV2/8-Spc5.12-µDys gene therapy in a relevant large-animal models of DMD and paves the way towards human clinical gene therapy using systemic peripheral vein administration of vector, and applicable to all DMD patients regardless of their genotype.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. Alternatively, dystrophic mesoangioblast-like cells were generated from iPS of DMD patients. These iPS-derived mesoangioblasts, constitute an essentially unlimited supply of stem/progenitor cells that could be genetically corrected using PB transposons expressing dystrophin. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

Human Gene TherapyVol. 27, No. 4 TributeA Tribute to George StamatoyannopoulosArun Srivastava, Mark A. Kay, Takis Athanasopoulos, Michael Angastiniotis, Achilles Anagnostopoulos, Garyfalia Karponi, Evangelia Yannaki, Leonard I. Zon, Carsten W. Lederer, Marios S. Phylactides, and Marina KleanthousArun SrivastavaSearch for more papers by this author, Mark A. KaySearch for more papers by this author, Takis AthanasopoulosSearch for more papers by this author, Michael AngastiniotisSearch for more papers by this author, Achilles AnagnostopoulosSearch for more papers by this author, Garyfalia KarponiSearch for more papers by this author, Evangelia YannakiSearch for more papers by this author, Leonard I. ZonSearch for more papers by this author, Carsten W. LedererSearch for more papers by this author, Marios S. PhylactidesSearch for more papers by this author, and Marina KleanthousSearch for more papers by this authorPublished Online:13 Apr 2016https://doi.org/10.1089/hum.2016.29025.gstAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 27Issue 4Apr 2016 InformationCopyright 2016, Mary Ann Liebert, Inc.To cite this article:Arun Srivastava, Mark A. Kay, Takis Athanasopoulos, Michael Angastiniotis, Achilles Anagnostopoulos, Garyfalia Karponi, Evangelia Yannaki, Leonard I. Zon, Carsten W. Lederer, Marios S. Phylactides, and Marina Kleanthous.A Tribute to George Stamatoyannopoulos.Human Gene Therapy.Apr 2016.280-286.http://doi.org/10.1089/hum.2016.29025.gstPublished in Volume: 27 Issue 4: April 13, 2016PDF download

O1 Regulation of genes by telomere length over long distances

Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies.

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.

In the 1950s, three groups made it their goal to determine the structure of DNA. The first group to start was at King's College London and was led by Maurice Wilkins and was later joined by Rosalind Franklin. Another group consisting of Francis Crick and James D. Watson was at Cambridge. A third group was at Caltech and was led by Linus Pauling. We have recently celebrated 60 years from the pioneering discovery of the Double Helix Structure (1953) announcing a series of articles in last year’s editorial of this Journal. In 2003, coinciding with the 50thanniversary of this momentous achievement in biology and the description of the DNA double helix was also the year that NHGRI celebrated the historic culmination of one of the most important scientific projects in history: the sequencing of the human genome.