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BACKGROUND AND AIMS: As cell and gene therapy (CGT) has grown in availability and scope, more unapproved regenerative medicine is being marketed to the public. It is essential that health care providers have sufficient knowledge and comfort to determine whether treatments are properly regulated and address these topics with patients. Due to the applicability of CGT to genetic disease, genetic counselors could be key in providing education and answering patients' questions about these topics. However, previous studies have focused only on physicians' knowledge and comfort with CGT and unapproved regenerative medicine. The purpose of this study was to assess genetic counselors' self-reported knowledge and comfort discussing these topics with patients and to explore what factors predict increased knowledge and comfort. METHODS: The authors designed an online survey distributed to genetic counselors who were part of the National Society of Genetic Counselors Student Research Program e-mail list. The survey addressed genetic counselors' demographics, practice experience with CGT, education about CGT, knowledge and comfort. RESULTS: The survey was completed by 144 genetic counselors. The best predictor of increased knowledge and comfort was experience discussing CGT in practice. In addition, those who worked at an institution at which CGT trials were offered had greater knowledge and comfort. However, most genetic counselors reported their knowledge was not sufficient to address questions from patients, and most had little-to-no knowledge or comfort determining whether a trial was properly regulated. There was no correlation between education and either knowledge or comfort; however, most participants desired more education about these topics. CONCLUSIONS: This study suggests that genetic counselors who (i) have experience with CGT in practice or (ii) work at institutions at which CGT trials are offered may have better knowledge regarding CGT. These results may help identify individuals and/or institutions in whom increasing knowledge regarding CGT could be beneficial. This is crucial as CGT becomes mainstream, leading to more widely marketed unapproved regenerative medicine. Several gaps in knowledge and comfort were identified, including participants' ability to determine whether a treatment is properly regulated. Further research is needed to better characterize the educational needs of genetic counselors surrounding these topics to address these gaps.
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Conselheiros , Aconselhamento Genético , Humanos , Aconselhamento Genético/métodos , Inquéritos e Questionários , Terapia Baseada em Transplante de Células e Tecidos , Medicina RegenerativaRESUMO
Workforce education and development are key cornerstones in advancing and maturing the Cell & Gene Therapy sector. A skilled worker shortage can significantly impact and delay progress as well as the quality of output for any developer, thereby negatively impacting a patient's access to life-saving treatments. Several roundtable discussions were held at the International Society for Cell & Gene Therapy (ISCT) 2023 Annual Meeting to dive deeper into the current state of workforce development and solutions to address this bottleneck. One roundtable discussion was co-hosted by the Alliance for Regenerative Medicine (ARM) and ISCT, which focused on the gap analysis provided for the United States Cell & Gene Therapy (CGT) sector, highlighting the lack of skilled workers in manufacturing and quality control. In this manuscript, the roundtable participants continue this conversation, review the roles and staffing requirements in both academic and industry as well as small and large company settings. The adoption of increased manufacturing automation is one promising solution to propel the sector forward. However, automation alone won't replace on-site staff, but will lower the bar to entry for a larger pool of people and require different training. This paper also addresses the workforce development and training paradigm shift as advanced manufacturing techniques are implemented, which will differ considerably based on the type of manufacturing efforts, thus emphasizing the need for a well-thought-out strategy to up-skill and re-skill the technical workforce to adapt to these advancements. Organizations such as ISCT and ARM have a role to play in propelling the field forward, providing awareness and education to stakeholders at all levels, as well as acting as a convener and participating as a key stakeholder in discussions and partnerships between academia and industry towards solutions for training the best personnel for CGT manufacturing. This scope includes novel digital tools and technologies to simplify training to increase access to new talent pools interested in careers in a rapidly advancing sector.
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Terapia Genética , Humanos , Medicina Regenerativa , Terapia Baseada em Transplante de Células e Tecidos , Estados Unidos , Recursos HumanosRESUMO
Adoptive cell therapy of tumor-infiltrating lymphocytes has shown promise for treatment of refractory melanoma and other solid malignancies; however, challenges to manufacturing have limited its widespread use. Traditional manufacturing efforts were lengthy, cumbersome and used open culture systems. We describe changes in testing and manufacturing that decreased the process cycle time, enhanced the robustness of critical quality attribute testing and facilitated a functionally closed system. These changes have enabled export of the manufacturing process to support multi-center clinical trials.
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Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura Celular por Lotes/tendências , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral , Controle de Qualidade , Sobrevivência Celular , Técnicas de Cocultura , Endotoxinas/análise , Humanos , Melanoma/patologia , Melanoma/terapia , MycoplasmaRESUMO
The GM.CD40L vaccine, which recruits and activates dendritic cells, migrates to lymph nodes, activating T cells and leading to systemic tumor cell killing. When combined with the CCL21 chemokine, which recruits T cells and enhances T-cell responses, additive effects have been demonstrated in non-small cell lung cancer mouse models. Here, we compared GM.CD40L versus GM.CD40L plus CCL21 (GM.CD40L.CCL21) in lung adenocarcinoma patients with ≥ 1 line of treatment. In this phase I/II randomized trial (NCT01433172), patients received intradermal vaccines every 14 days (3 doses) and then monthly (3 doses). A two-stage minimax design was used. During phase I, no dose-limiting toxicities were shown in three patients who received GM.CD40L.CCL21. During phase II, of evaluable patients, 5/33 patients (15.2%) randomized for GM.DCD40L (p = .023) and 3/32 patients (9.4%) randomized for GM.DCD40L.CCL21 (p = .20) showed 6-month progression-free survival. Median overall survival was 9.3 versus 9.5 months with GM.DCD40L versus GM.DCD40L.CCL21 (95% CI 0.70-2.25; p = .44). For GM.CD40L versus GM.CD40L.CCL21, the most common treatment-related adverse events (TRAEs) were grade 1/2 injection site reaction (51.4% versus 61.1%) and grade 1/2 fatigue (35.1% versus 47.2%). Grade 1 immune-mediated TRAEs were isolated to skin. No patients showed evidence of pseudo-progression or immune-related TRAEs of grade 1 or greater of pneumonitis, endocrinopathy, or colitis, and none discontinued treatment due to toxicity. Although we found no significant associations between vaccine immunogenicity and outcomes, in limited biopsies, one patient treated with GMCD40L.CCL21 displayed abundant tumor-infiltrating lymphocytes. This possible effectiveness warrants further investigation of GM.CD40L in combination approaches.
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Adenocarcinoma/terapia , Ligante de CD40/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/terapia , Quimiocina CCL21/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Imunoterapia , Adenocarcinoma/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de SobrevidaRESUMO
Host innate-immune responses are tailored by cell type to control and eradicate specific infectious agents. For example, an acute RNA virus infection can result in high-level expression of type 1 IFNs by both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs), but whereas cDCs preferentially use RIG-I-like receptor (RLR) signaling to produce type 1 IFNs, pDCs predominantly use TLRs to induce these cytokines. We previously found that the IκB kinase ß (IKKß)/NF-κB pathway regulates early IFN-ß expression, but not the magnitude of type 1 IFN expression following RLR engagement. In this study, we use IKKß inhibition and mice deficient in IKKß or canonical NF-κB subunits (p50, RelA/p65, and cRel) to demonstrate that the IKKß/NF-κB axis is critical for virus-induced type 1 IFN expression in pDCs, but not in cDCs. We also reveal a crucial and more general requirement for IKKß/NF-κB in TLR- but not RLR-induced expression of type 1 IFNs and inflammatory cytokines. Together, these findings reveal a previously unappreciated specificity of the IKKß/NF-κB signaling axis in regulation of antimicrobial responses by different classes of pattern recognition receptors, and therefore by individual cell types reliant on particular pattern recognition receptors for their innate-immune transcriptional responses.
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Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Quinase I-kappa B/imunologia , Interferon Tipo I/imunologia , NF-kappa B/imunologia , Plasmócitos/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Animais , Regulação da Expressão Gênica/genética , Quinase I-kappa B/genética , Interferon Tipo I/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Receptores de Superfície Celular , Transdução de Sinais/genética , Receptores Toll-Like/genéticaRESUMO
In the accepted model of T-cell activation, parallel signal-transduction pathways activate the transcription factors NF-κB, NFAT, and AP-1 to drive clonal expansion of T cells in response to Ag. Genome-wide transcriptional profiling following Ag-induced CD8(+) T-cell activation in C57BL/6 mouse T cells revealed that genes regulated by NFAT were also reduced in the absence of NF-κB p50 and cRel subunits. Importantly, p50(-/-) cRel(-/-) CD8(+) T cells had significantly diminished NFAT and AP-1 activation compared with WT or PKCθ(-/-) CD8(+) T cells. Attenuated NFAT activation after TCR engagement was associated with reduced calcium influx, PLCγ and Zap70 activation. Interestingly, pharmacological bypass of PLCγ-regulated pathways largely rescued p50(-/-) cRel(-/-) T-cell proliferative defects. These results indicate a crucial and unexpected requirement for NF-κB p50 and cRel subunits in proximal TCR signaling and calcium responses. They further suggest that key defects in T cells in the absence of NF-κB pathway components may be due to impaired proximal T-cell signaling.
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Linfócitos T CD8-Positivos/imunologia , Sinalização do Cálcio/imunologia , Subunidade p50 de NF-kappa B/imunologia , Fatores de Transcrição NFATC/imunologia , Proteínas Proto-Oncogênicas c-rel/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Sinalização do Cálcio/genética , Proliferação de Células/fisiologia , Camundongos , Camundongos Knockout , Subunidade p50 de NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Proteínas Proto-Oncogênicas c-rel/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
Microbial adjuvants in vaccines activate key transcription factors, including NF-κB and interferon response factors (IRFs). However, the individual role of these transcription factor pathways in promoting adaptive immunity by adjuvants is not clear. It is widely believed that induction of a strong inflammatory response potentiates an adaptive immune response. In this study, we sought to determine whether activation of the pro-inflammatory inhibitor of κB kinase ß (IKKß) canonical NF-κB pathway promoted vaccine-induced immune responses. An adenovirus expressing constitutively activated IKKß (AdIKK) induced robust DC maturation and high expression of key cytokines compared with a control virus. In vivo, AdIKK triggered rapid inflammation after pulmonary infection, increased leukocyte entry into draining LNs, and enhanced early antibody and T-cell responses. Notably, AdIKK did not influence the overall magnitude of the adaptive immune response. These results indicate that induction of inflammation by IKKß/NFκB in this setting impacts the kinetics but not the magnitude of adaptive immune responses. These findings therefore help define the individual role of a key pathway induced by vaccine adjuvants in promoting adaptive immunity.
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Imunidade Adaptativa/imunologia , Adenoviridae/imunologia , Quinase I-kappa B/imunologia , Imunização/normas , Pulmão/imunologia , NF-kappa B/imunologia , Adenoviridae/genética , Adjuvantes Imunológicos/farmacologia , Animais , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos EspecíficosRESUMO
RNA virus infection results in expression of type 1 IFNs, especially IFN-alpha/beta, which play a crucial role in host antivirus responses. Type 1 IFNs are induced in a cell type-specific manner through TLR and RIG-I-like receptor pathways, both of which activate IFN regulatory factors (IRFs) and NF-kappaB transcription factors. Although NF-kappaB activation and association with the IFN-beta promoter after RNA virus infection is well documented, our previous work showed that, surprisingly, NF-kappaB is not essential for IFN-beta gene expression. Thus, the actual function of NF-kappaB in IFN-beta expression and virus replication is not clear. In this study, we found Newcastle disease virus and vesicular stomatitis virus replication is enhanced in mouse embryonic fibroblasts (MEFs) lacking the NF-kappaB RelA subunit. Increased virus replication was traced to a specific requirement for RelA in early virus-induced IFN-beta expression. At these time points, when IFN-beta expression is ~100-fold less than peak levels, impaired IFN-beta production delayed IFN-induced gene expression, resulting in increased virus replication in RelA(-/-) MEFs. Importantly, our results show that RelA requirement is crucial only when IRF3 activation is low. Thus, high levels of activated IRF3 expression are sufficient for induction of IFN-beta in RelA(-/-) MEFs, transcriptional synergism with the coactivator CREB-binding protein, and rescue of susceptibility to virus. Together, these findings indicate that NF-kappaB RelA is not crucial for regulating overall IFN-beta production, as previously believed; instead, RelA is specifically required only during a key early phase after virus infection, which substantially impacts the host response to virus infection.
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Interferon beta/biossíntese , Subunidade p50 de NF-kappa B/fisiologia , Vírus da Doença de Newcastle/imunologia , Fator de Transcrição RelA/fisiologia , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral/imunologia , Animais , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Interferon beta/genética , Camundongos , Camundongos Knockout , Vírus da Doença de Newcastle/fisiologia , Fatores de Tempo , Fator de Transcrição RelA/deficiência , Fator de Transcrição RelA/genética , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/genéticaRESUMO
Dendritic cells play a key role in activation of the immune system as potent antigen-presenting cells. This pivotal position, along with the ability to generate dendritic cells from monocytes and ready uptake of antigen, makes them an intriguing vehicle for immunotherapy for a variety of indications. Since the first reported trial using dendritic cells in 1995, they have been used in trials all over the world for a plethora of indications. Monocyte-derived dendritic cells are generated from whole blood or apheresis products by culturing enriched monocytes in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). A variety of methods can be used for enrichment of monocytes for generation of clinical-grade dendritic cells and are summarized herein.
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NF-κB is constitutively activated in many cancer types and is a potential key mediator of tumor-associated inflammation, tumor growth, and metastasis. We investigated the role of cancer cell NF-κB activity in T cell-mediated antitumor responses. In tumors rendered immunogenic by model antigen expression or following administration of antitumor vaccines, we found that high NF-κB activity leads to tumor rejection and/or growth suppression in mice. Using a global RNA expression microarray, we demonstrated that NF-κB enhanced expression of several T cell chemokines, including Ccl2, and decreased CCL2 expression was associated with enhanced tumor growth in a mouse lung cancer model. To investigate NF-κB function in human lung tumors, we identified a gene expression signature in human lung adenocarcinoma cell lines that was associated with NF-κB activity level. In patient tumor samples, overall lung tumor NF-κB activity was strongly associated with T cell infiltration but not with cancer cell proliferation. These results therefore indicate that NF-κB activity mediates immune surveillance and promotes antitumor T cell responses in both murine and human lung cancer.
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Adenocarcinoma/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Neoplasias Pulmonares/imunologia , NF-kappa B/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Animais , Carcinoma Pulmonar de Lewis/metabolismo , Linhagem Celular Tumoral , Quimiocinas/genética , Quimiocinas/metabolismo , Expressão Gênica/imunologia , Rejeição de Enxerto , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Fatores Imunológicos/genética , Fatores Imunológicos/metabolismo , Vigilância Imunológica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neutrófilos/imunologia , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Linfócitos T/metabolismo , Transcriptoma/imunologiaRESUMO
When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform theta (PKCtheta), a key regulator of TCR signaling. In contrast, PKCtheta was required for alloreactivity and GVHD induction. Furthermore, absence of PKCtheta raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCtheta-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCtheta is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.