Global collaboration is the cornerstone of scientific advancement.? Frontiers in Medicine has organized a series of special edition Research Topics, with the goal of highlighting the latest advancements in Gene and Cell Therapy across the globe, showcasing the academic excellence and high-quality work of internationally recognized researchers.? These collections aim to shed light on the recent progress made across the entire breadth of the Gene and Cell Therapy field, and reflect on the future challenges faced by researchers across borders. This topic will focus specifically on contributions to the field from European researchers.

Centre for Neuroscience and Cell Biology, University of Coimbra

University of Algarve

University of Warwick

Exploring contributions from European gene and cell therapy researchers for global success

. In the last decade, there has been an increased interest in cell and gene based therapies for a range of medical conditions. This has led to the development of legislation specifically focused on these therapies, however there are numerous barriers that must be overcome before they can be adopted on a mainstream level. Riboswitches have been studied for their potential use in gene therapy, and this research has revealed significant differences in their function. Additionally, this research provides important aspects on optimizing expression cassette designs to benefit further research and development. <ul> <li>Adeno-associated viruses (AAVs) have been studied for their antiangiogenic and antitumoral effects, and their combined use with paclitaxel and carboplatin chemotherapy in an ovarian cancer mouse model.</li> <li>CRISPR/Cas9-based gene editing approaches have been studied for their potential use in the treatment of junctional epidermolysis bullosa (JEB) caused by mutations in the COL17A1 gene.</li> <li>Cell and Gene Therapy Products (CGT) require a risk-based approach to develop a sound regulatory strategy for clinical development and registration.</li> <li>Adeno-associated viruses (AAVs) have been studied in 136 clinical trials over two decades, and optimal dosing regimes remain elusive.</li> </ul>

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Frontiers in Medicine

Gene and Cell Therapy

Global Excellence in Gene and Cell Therapy: Europe

Gene therapy changes protein expression in cells through the insertion of genes and/or gene expression-modulating tools, including gene editing. It can be used in the treatment of a variety of diseases, including genetic disorders, by reducing the levels of disease-causing proteins or by increasing the expression of missing or protective proteins. Cell therapy by the transplantation of cells or tissues aims to repair, regenerate, or replace a specific cell population and/or tissue to reestablish the physiological function performed by the cells/tissues to replace. In this line, Global Excellence in the Gene and Cell Therapy: Europe Research Topic was launched with the goal of highlighting the latest advancements in Gene and Cell Therapy field and reflecting on the future challenges faced by researchers across Europe.Six papers were accepted and published for this Research Topic, from which three describe original research data, one is a systematic review, one is a narrative paper, and the last one is a perspective article.The systematic review by <a href="https://doi.org/10.3389/fmed.2021.809118">Au et al.</a>, Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings addressed the use of Adeno-associated viruses (AAVs) as gene delivery vehicles to drive long-term transgene expression. This meta-analysis of two decades of AAV application in gene therapy, in 136 clinical trials, provided an up-to-date overview of the use of AAVs in clinical trials, while evaluating the major challenges in the AAVs' translatability into clinical practice. These comprise restricted gene packaging capacities, off-target transduction, and immunogenicity. The authors also pointed out some aspects in this research field that should be further investigated, namely the development of optimal dosing regimens based on dose-dependent toxicity and efficacy studies, the engineering of new capsid variants to improve transduction and reduce immunogenicity, and the use of better-suited promoters. Accordingly, this paper provides an overview of AAV-mediated gene therapy.In their original research paper COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa, <a href="https://doi.org/10.3389/fmed.2022.976604">Petković et al.</a> reported a gene editing approach to correct a frameshift mutation within the COL17A1 gene associated with junctional epidermolysis bullosa (JEB). In this work, type XVII collagen (C17)-deficient JEB keratinocytes were edited with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) homology-directed repair (HDR) template. Next-generation sequencing of RNP-nucleofected keratinocytes demonstrated an HDR efficiency of approximately 38% with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and a ssODN template. Moreover, gene-corrected JEB keratinocytes upon differentiation into skin equivalents displayed enhanced adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone. Overall, this work describes a promising CRISPR/Cas9-based gene editing strategy.<a href="https://doi.org/10.3389/fmed.2022.1018208">Kujala et al.</a> report in their original research paper AAV8-mediated sVEGFR2 and sVEGFR3 gene therapy combined with chemotherapy reduces the growth and microvasculature of human ovarian cancer and prolongs the survival in mice the antiangiogenic and antitumoral effects on ovarian cancer (OVCA) of adeno-associated virus 8 (AAV8)-mediated expression of soluble VEGF receptors (sVEGFRs) sVEGFR2 and sVEGFR3 combined with the chemotherapy drugs paclitaxel and carboplatin. Mice inoculated with human OVCA cells were tested with: (i) AAV8-CMV and chemotherapy, (ii) AAV8-sVEGFR2, (iii) AAV8-sVEGFR3, (iv) AAV8-sVEGFR2 and AAV8-sVEGFR3, and (v) AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy. Authors reported that the therapeutic combination of AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy significantly limited intratumoral angiogenesis and tumor growth. Therefore, this work demonstrates the potential synergistic benefits of combining antiangiogenic therapy and chemotherapy in human OVCA treatment.The original research work by <a href="https://doi.org/10.3389/fmed.2022.1052318">Eriksson et al.</a> Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy investigated a method to temporally regulate therapeutic gene expression with Riboswitches. Several tetracycline and toyocamycin-inducible ON-riboswitches were tested. The tetracycline-dependent K19 riboswitch displayed the best expression control of several transgenes. The gene expression induction was 6- to 10-fold, reversible, dose-dependent, and took place within hours upon tetracycline administration. The authors further optimized the gene cassette to control the expression of VEGF-B, a gene with therapeutic potential for cardiovascular diseases. They found that the riboswitch function was promoter-independent, the use of two or three riboswitches simultaneously reduced leakiness and improved the temporal dynamic range, and a linker sequence between the riboswitches improves their functionality. Therefore, this study indicates that Riboswitch-regulated transgene expression is a promising strategy for safe, dose-controlled gene therapy and provides important insights into the optimization of this strategy.The review Barriers to Treg therapy in Europe: from production to regulation (<a href="https://doi.org/10.3389/fmed.2023.1090721">Hennessy et al.</a>) analyzes some critical aspects in the development of advanced therapy medicinal products (ATMPs) such as regulatory T cells (Tregs). Several parameters required to ensure the safety, efficacy, and quality of these medicinal products are discussed, namely the source of cells to be employed, the methods through which the cells are isolated and expanded, and the cells' storage. Regulatory issues are also analyzed including the mandatory compliance with Good Manufacturing Procedure (GMP) guidelines and the challenges of its implementation. Moreover, an overview of the currently approved ATMPs in Europe and the costs of the development of this type of medicinal products is provided.<a href="https://doi.org/10.3389/fmed.2022.855100">Salazar-Fontana</a> contributed with the perspective article A Regulatory Risk-Based Approach to ATMP/CGT Development: Integrating Scientific Challenges With Current Regulatory Expectations, providing an overview on the European Union and United States Regulatory Landscape for ATMP/Cell and Gene Therapy (CGT) Products. Some aspects of the ATMP/CGT development and the guidelines established by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) on the quality, non-clinical and clinical considerations were reviewed. The paper discusses, among other subjects, the timelines for product development and the review of the license application. Thus, the accelerated drug development processes Orphan Drug Designation (ODD), EU PRIME, and US RMAT were considered. Moreover, the EMA and FDA's different approach regarding the regulatory requirements of Genetically Modified Organisms (GMO) for ATMP/ CGT was also addressed.Overall, the articles in the present Research Topic advanced on the development of new gene delivery and gene editing tools and provided an overview of the Regulatory Landscape for ATMPs.LM wrote the manuscript. JB and CN revised the manuscript. All authors approved the submitted version.The editors would like to thank all the authors and reviewers that participated in the Research Topic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Editorial: Global excellence in gene and cell therapy: Europe

There has been an increased interest in cell based therapies for a range of medical conditions in the last decade. This explosion in novel therapeutics research has led to the development of legislation specifically focused on cell and gene based therapies. In Europe, the European medicines agency (EMA) designates any medicines for human use which are based on genes, tissues, or cells as advanced therapy medicinal products or advanced therapy medicinal products (ATMPs). In this article we discuss the hurdles to widespread adoption of ATMPs in Europe, with a focus on regulatory T cells (Tregs). There are numerous barriers which must be overcome before mainstream adoption of Treg therapy becomes a reality. The source of the cells, whether to use autologous or allogenic cells, and the methods through which they are isolated and expanded, must all meet strict good manufacturing practice (GMP) standards to allow use of the products in humans. GMP compliance is costly, with the equipment and reagents providing a significant cost barrier and requiring specialized facilities and personnel. Conforming to the regulations set centrally by the EMA is difficult, and the different interpretations of the regulations across the various member states further complicates the regulatory approval process. The end products then require a complex and robust distribution network to ensure timely delivery of potentially life saving treatments to patients. In a European market whose logistics networks have been hammered by COVID and Brexit, ensuring rapid and reliable delivery systems is a more complex task than ever. In this article we will examine the impact of these barriers on the development and adoption of Tregs in Europe, and potential approaches which could facilitate more widespread use of Tregs, instead of its current concentration in a few very specialized centers.

There has been an increased interest in cell based therapies for a range of medical conditions in the last decade. This explosion in novel therapeutics research has led to the development of legislation specifically focused on cell and gene based therapies. In Europe, the European medicines agency (EMA) designates any medicines for human use which are based on genes, tissues, or cells as advanced therapy medicinal products or advanced therapy medicinal products (ATMPs). In this article we discuss the hurdles to widespread adoption of ATMPs in Europe, with a focus on regulatory T cells (Tregs). There are numerous barriers which must be overcome before mainstream adoption of Treg therapy becomes a reality. The source of the cells, whether to use autologous or allogenic cells, and the methods through which they are isolated and expanded, must all meet strict good manufacturing practice (GMP) standards to allow use of the products in humans. GMP compliance is costly, with the equipment and reagents providing a significant cost barrier and requiring specialized facilities and personnel. Conforming to the regulations set centrally by the EMA is difficult, and the different interpretations of the regulations across the various member states further complicates the regulatory approval process. The end products then require a complex and robust distribution network to ensure timely delivery of potentially life saving treatments to patients. In a European market whose logistics networks have been hammered by COVID and Brexit, ensuring rapid and reliable delivery systems is a more complex task than ever. In this article we will examine the impact of these barriers on the development and adoption of Tregs in Europe, and potential approaches which could facilitate more widespread use of Tregs, instead of its current concentration in a few very specialized centers.

Barriers to Treg therapy in Europe: From production to regulation

Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B. Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3′ end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.

Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B. Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3′ end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.

Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy

BackgroundVascular endothelial growth factors (VEGFs) are major regulators of intratumoral angiogenesis in ovarian cancer (OVCA). Overexpression of VEGFs is associated with increased tumor growth and metastatic tendency and VEGF-targeting therapies are thus considered as potential treatments for OVCA. Here, we examined the antiangiogenic and antitumoral effects on OVCA of adeno-associated virus 8 (AAV8)-mediated expression of soluble VEGF receptors (sVEGFRs) sVEGFR2 and sVEGFR3 together with paclitaxel and carboplatin chemotherapy.</sec>Materials and methodsImmunodeficient mice were inoculated with human OVCA cell line SKOV-3m. Development of tumors was confirmed with magnetic resonance imaging (MRI) and mice were treated with gene therapy and paclitaxel and carboplatin chemotherapy. The study groups included (I) non-treated control group, (II) blank control vector AAV8-CMV, (III) AAV8-CMV with chemotherapy, (IV) AAV8-sVEGFR2, (V) AAV8-sVEGFR3, (VI) AAV8-sVEGFR2 and AAV8-sVEGFR3, and (VII) AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy. Antiangiogenic and antitumoral effects were evaluated with immunohistochemical stainings and serial MRI.</sec>ResultsReduced intratumoral angiogenesis was observed in all antiangiogenic gene therapy groups. The combined use of AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy suppressed ascites fluid formation and tumor growth, thus improving the overall survival of mice. Antitumoral effect was mainly caused by AAV8-sVEGFR2 while the benefits of AAV8-sVEGFR3 and chemotherapy were less prominent.</sec>ConclusionCombined use of the AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy reduces intratumoral angiogenesis and tumor growth in OVCA mouse model. Results provide preclinical proof-of-concept for the use of soluble decoy VEGFRs and especially the AAV8-sVEGFR2 in the treatment of OVCA.</sec>

Background: Vascular endothelial growth factors (VEGFs) are major regulators of intratumoral angiogenesis in ovarian cancer (OVCA). Overexpression of VEGFs is associated with increased tumor growth and metastatic tendency and VEGF-targeting therapies are thus considered as potential treatments for OVCA. Here, we examined the antiangiogenic and antitumoral effects on OVCA of adeno-associated virus 8 (AAV8)-mediated expression of soluble VEGF receptors (sVEGFRs) sVEGFR2 and sVEGFR3 together with paclitaxel and carboplatin chemotherapy.Materials and methods: Immunodeficient mice were inoculated with human OVCA cell line SKOV-3m. Development of tumors was confirmed with magnetic resonance imaging (MRI) and mice were treated with gene therapy and paclitaxel and carboplatin chemotherapy. The study groups included (I) non-treated control group, (II) blank control vector AAV8-CMV, (III) AAV8-CMV with chemotherapy, (IV) AAV8-sVEGFR2, (V) AAV8-sVEGFR3, (VI) AAV8-sVEGFR2 and AAV8-sVEGFR3, and (VII) AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy. Antiangiogenic and antitumoral effects were evaluated with immunohistochemical stainings and serial MRI.Results: Reduced intratumoral angiogenesis was observed in all antiangiogenic gene therapy groups. The combined use of AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy suppressed ascites fluid formation and tumor growth, thus improving the overall survival of mice. Antitumoral effect was mainly caused by AAV8-sVEGFR2 while the benefits of AAV8-sVEGFR3 and chemotherapy were less prominent.Conclusion: Combined use of the AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy reduces intratumoral angiogenesis and tumor growth in OVCA mouse model. Results provide preclinical proof-of-concept for the use of soluble decoy VEGFRs and especially the AAV8-sVEGFR2 in the treatment of OVCA.

AAV8-mediated sVEGFR2 and sVEGFR3 gene therapy combined with chemotherapy reduces the growth and microvasculature of human ovarian cancer and prolongs the survival in mice

BackgroundEpidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in COL17A1 in the treatment of junctional epidermolysis bullosa (JEB).</sec>MethodsIn this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the COL17A1 gene.</sec>ResultsAs analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of ∼38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents.</sec>ConclusionHere we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR/Cas9-based gene editing approach for JEB.</sec>

Background: Epidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in COL17A1 in the treatment of junctional epidermolysis bullosa (JEB).Methods: In this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the COL17A1 gene.Results: As analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of ∼38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents.Conclusion: Here we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR/Cas9-based gene editing approach for JEB.

COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa

Cell and Gene Therapy Products (CGT), regulated as Advanced Therapy Medicinal Products (ATMP) in the European Union (EU), represent a novel and varied group of biotherapeutics developed to treat specific conditions for which there are limited or no effective treatments. The novelty and complexity of this product modality demands a regulatory risk-based approach to define a sound development plan, particularly, as most developers aim to target more than one market area simultaneously for clinical development and registration. This regulatory strategy should be built on solid scientific data that also addresses general regulatory recommendations to enable a benefit:risk analysis that is aligned with the particularities of each CGT product. This risk-based approach is especially helpful when no detailed product-specific regulatory guidelines are available. The goal of this article is to orient developers on how to build a combined EU/US regulatory strategy through the assessment of commonly understood quality (CMC), non-clinical, and clinical regulatory risks faced by ATMP/CGT.

Cell and Gene Therapy Products (CGT), regulated as Advanced Therapy Medicinal Products (ATMP) in the European Union (EU), represent a novel and varied group of biotherapeutics developed to treat specific conditions for which there are limited or no effective treatments. The novelty and complexity of this product modality demands a regulatory risk-based approach to define a sound development plan, particularly, as most developers aim to target more than one market area simultaneously for clinical development and registration. This regulatory strategy should be built on solid scientific data that also addresses general regulatory recommendations to enable a benefit:risk analysis that is aligned with the particularities of each CGT product. This risk-based approach is especially helpful when no detailed product-specific regulatory guidelines are available. The goal of this article is to orient developers on how to build a combined EU/US regulatory strategy through the assessment of commonly understood quality (CMC), non-clinical, and clinical regulatory risks faced by ATMP/CGT.

A Regulatory Risk-Based Approach to ATMP/CGT Development: Integrating Scientific Challenges With Current Regulatory Expectations

Adeno-associated viruses (AAVs) are the safest and most effective gene delivery vehicles to drive long-term transgene expression in gene therapy. While animal studies have shown promising results, the translatability of AAVs into clinical settings has been partly limited due to their restricted gene packaging capacities, off-target transduction, and immunogenicity. In this study, we analysed over two decades of AAV applications, in 136 clinical trials. This meta-analysis aims to provide an up-to-date overview of the use and successes of AAVs in clinical trials, while evaluating the approaches used to address the above challenges. First, this study reveals that the speed of novel AAV development has varied between therapeutic areas, with particular room for improvement in Central Nervous System disorders, where development has been slow. Second, the lack of dose-dependent toxicity and efficacy data indicates that optimal dosing regimes remain elusive. Third, more clinical data on the effectiveness of various immune-modulation strategies and gene editing approaches are required to direct future research and to accelerate the translation of AAV-mediated gene therapy into human applications.

Adeno-associated viruses (AAVs) are the safest and most effective gene delivery vehicles to drive long-term transgene expression in gene therapy. While animal studies have shown promising results, the translatability of AAVs into clinical settings has been partly limited due to their restricted gene packaging capacities, off-target transduction, and immunogenicity. In this study, we analysed over two decades of AAV applications, in 136 clinical trials. This meta-analysis aims to provide an up-to-date overview of the use and successes of AAVs in clinical trials, while evaluating the approaches used to address the above challenges. First, this study reveals that the speed of novel AAV development has varied between therapeutic areas, with particular room for improvement in Central Nervous System disorders, where development has been slow. Second, the lack of dose-dependent toxicity and efficacy data indicates that optimal dosing regimes remain elusive. Third, more clinical data on the effectiveness of various immune-modulation strategies and gene editing approaches are required to direct future research and to accelerate the translation of AAV-mediated gene therapy into human applications.