In silico and in vivo analysis reveal impact of c-Myc tag in FMC63 scFv-CD19 protein interface and CAR-T cell efficacy.

Anti-CD19 CAR-T cell therapy represents a breakthrough in the treatment of B-cell malignancies, and it is expected that this therapy modality will soon cover a range of solid tumors as well. Therefore, a universal cheap and sensitive method to detect CAR expression is of foremost importance. One possibility is the use of epitope tags such as c-Myc, HA or FLAG tags attached to the CAR extracellular domain, however, it is important to determine whether these tags can influence binding of the CAR with its target molecule. Here, we conducted in-silico structural modelling of an FMC63-based anti-CD19 single-chain variable fragment (scFv) with and without a c-Myc peptide tag added to the N-terminus portion and performed molecular dynamics simulation of the scFv with the CD19 target. We show that the c-Myc tag presence in the N-terminus portion does not affect the scFv's structural equilibrium and grants more stability to the scFv. However, intermolecular interaction potential (IIP) analysis reveals that the tag can approximate the complementarity-determining regions (CDRs) present in the scFv and cause steric impediment, potentially disturbing interaction with the CD19 protein. We then tested this possibility with CAR-T cells generated from human donors in a Nalm-6 leukemia model, showing that CAR-T cells with the c-Myc tag have overall worse antitumor activity, which was also observed when the tag was added to the C-terminus position. Ultimately, our results suggest that tag addition is an important aspect of CAR design and can influence CAR-T cell function, therefore its use should be carefully considered.

Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells.

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

CRISPR/Cas-9 vector system: targets UL-39 and inhibits Simplexvirus humanalpha1 (HSV-1) replication in vitro.

Simplexvirus humanalpha1 (HSV-1) affects approximately 67% of the world's population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into the plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyses revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas 9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.

Implementation of a gene therapy education initiative by the ASGCT and Muhimbili University of Health and Allied Sciences.

There has been rapid growth in gene therapy development with an expanding list of approved clinical products. Several therapies are particularly relevant to patients in low- and middle-income countries. Moreover, investing in research and manufacturing presents an opportunity for economic development. To increase awareness of gene therapy, the American Society of Gene and Cell Therapy partnered with the Muhimbili University of Health and Allied Sciences, Tanzania, to create a certificate-bearing course. The goal was to provide faculty teaching in graduate and medical schools with the tools needed to add gene therapy to the university curriculum. The first virtual course was held in October of 2022, and 45 individuals from 9 countries in Africa completed the training. The content was new to approximately two-thirds of participants, with the remaining third indicating that the course increased their knowledge base. The program was well received and will be adapted for other under-resourced regions.

The CRISPR/Cas System in Human Cancer.

The use of CRISPR as a genetic editing tool modified the oncology field from its basic to applied research for opening a simple, fast, and cheaper way to manipulate the genome. This chapter reviews some of the major uses of this technique for in vitro- and in vivo-based biological screenings, for cellular and animal model generation, and new derivative tools applied to cancer research. CRISPR has opened new frontiers increasing the knowledge about cancer, pointing to new solutions to overcome several challenges to better understand the disease and design better treatments.

Oral Lichen Planus and Oral Squamous Cell Carcinoma share key oncogenic signatures.

To investigate similarities in the gene profile of Oral Lichen Planus and Oral Squamous Cell Carcinoma that may justify a carcinogenic potential, we analyzed the gene expression signatures of Oral Lichen Planus and Oral Squamous Cell Carcinoma in early and advanced stages. Based on gene expression data from public databases, we used a bioinformatics approach to compare expression profiles, estimate immune infiltrate composition, identify differentially and co-expressed genes, and propose putative therapeutic targets and associated drugs. Our results revealed gene expression patterns related to processes of keratinization, keratinocyte differentiation, cell proliferation and immune response in common between Oral Lichen Planus and early and advanced Oral Squamous Cell Carcinoma, with the cornified envelope formation and antigen processing cross-presentation pathways in common between Oral Lichen Planus and early Oral Squamous Cell Carcinoma. Together, these results reveal that key tumor suppressors and oncogenes such as PI3, SPRR1B and KRT17, as well as genes associated with different immune processes such as CXCL13, HIF1A and IL1B are dysregulated in OLP.

Gene therapy access: Global challenges, opportunities, and views from Brazil, South Africa, and India.

Gene and cell therapies for a variety of life-limiting illnesses are under investigation, and a small number of commercial products have successfully obtained regulatory approval. The cost of treatment is high, and clinical studies evaluating safety and efficacy are performed predominately in high-income countries. We reviewed the current status of gene and cell therapies in low- and middle-income countries and highlighted the need and current barriers to access. The state of product development in Brazil, South Africa, and India is discussed, including lessons learned from American Society of Gene and Cell Therapy (ASGCT)-sponsored virtual symposia in each of these countries.

The ambiguous role of obesity in oncology by promoting cancer but boosting antitumor immunotherapy.

Obesity is nowadays considered a pandemic which prevalence's has been steadily increasingly in western countries. It is a dynamic, complex, and multifactorial disease which propitiates the development of several metabolic and cardiovascular diseases, as well as cancer. Excessive adipose tissue has been causally related to cancer progression and is a preventable risk factor for overall and cancer-specific survival, associated with poor prognosis in cancer patients. The onset of obesity features a state of chronic low-grade inflammation and secretion of a diversity of adipocyte-derived molecules (adipokines, cytokines, hormones), responsible for altering the metabolic, inflammatory, and immune landscape. The crosstalk between adipocytes and tumor cells fuels the tumor microenvironment with pro-inflammatory factors, promoting tissue injury, mutagenesis, invasion, and metastasis. Although classically established as a risk factor for cancer and treatment toxicity, recent evidence suggests mild obesity is related to better outcomes, with obese cancer patients showing better responses to treatment when compared to lean cancer patients. This phenomenon is termed obesity paradox and has been reported in different types and stages of cancer. The mechanisms underlying this paradoxical relationship between obesity and cancer are still not fully described but point to systemic alterations in metabolic fitness and modulation of the tumor microenvironment by obesity-associated molecules. Obesity impacts the response to cancer treatments, such as chemotherapy and immunotherapy, and has been reported as having a positive association with immune checkpoint therapy. In this review, we discuss obesity's association to inflammation and cancer, also highlighting potential physiological and biological mechanisms underlying this association, hoping to clarify the existence and impact of obesity paradox in cancer development and treatment.

The prevalence and prognostic impact of tumor-infiltrating lymphocytes in uterine carcinosarcoma.

OBJECTIVE: To examine the prevalence and prognostic role of tumor microenvironment (TME) markers in uterine carcinosarcoma (UCS) through immunohistochemical characterization. METHODS: The internal database of our institution was queried out for women with UCS who underwent surgery and thereafter postoperative chemotherapy with carboplatin and paclitaxel between January 2012 and December 2017. Tissue microarrays containing surgical samples of UCS from 57 women were assessed by immunohistochemistry for CD3, CD4, CD8, FOXP3, PD-1, PD-L1, and PD-L2. RESULTS: The mean age was 65.3 years (range, 49 to 79 years). For the epithelial component (E), CD3_E and CD4_E were highly expressed in 38 (66.7%) and in 40 (70.1%) patients, respectively, and were significantly associated with more advanced stages (p = 0.038 and p = 0.025, respectively). CD8_E was highly expressed in 42 (73.7%) patients, FOXP3_E 16 (28.1%), PD-1_E 35 (61.4%), PD-L1_E 27 (47.4%) and PD-L2_E 39 (68.4%). For the sarcomatous component (S), the prevalence of high expression was: CD3_S 6 (10.5%), CD4_S 20 (35.1%), CD8_S 44 (77.2%), FOXP3_S 8 (14%), PD-1_S 14 (24.6%), PD-L1_S 14 (24.6%) and PD-L2_S 8 (14%). By multivariate analysis, the CD8/FOXP3_S ratio (p = 0.026), CD4_E (p = 0.010), PD-L1_E (p = 0.013) and PD-L1_S (p = 0.008) markers significantly influenced progression-free survival. CD4/FOXP3_S ratio (p = 0.043), PD-1_E (p = 0.011), PD-L1_E (p = 0.036) and PD-L1_S (p = 0.028) had a significant association with overall survival. CONCLUSION: Some differences in UCS clinical outcomes may be due to the subtype of TILs and PD-1/PD-L1 axis immune checkpoint signaling.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VII. Present and future of technologies for production of CAR cell therapies.

Chimeric Antigen Receptor T (CAR-T) cells are certainly an important therapy for patients with relapsed and/or refractory hematologic malignancies. Currently, there are five CAR-T cell products approved by the FDA but several research groups and/or biopharmaceutical companies are encouraged to develop new products based on CAR cells using T or other cell types. Production of CAR cells requires intensive work from the basic, pre-clinical to translational levels, aiming to overcome technical difficulties and failure in the production. At least five key common steps are needed for the manipulation of T-lymphocytes (or other cells), such as: cell type selection, activation, gene delivery, cell expansion and final product formulation. However, reproducible manufacturing of high-quality clinical-grade CAR cell products is still required to apply this technology to a greater number of patients. This chapter will discuss the present and future development of new CAR designs that are safer and more effective to improve this therapy, achieving more selective killing of malignant cells and less toxicity to be applied in the clinical setting.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. VIII: CAR-T cells: preclinical development - Safety and efficacy evaluation.

Currently, there are four CAR-T products commercially available on the market. CAR-T cells have shown high remission rates and they represent an effective treatment option for patients with resistant or refractory B cell malignancies. Approval of these cell therapy products came after an extended period of preclinical evaluation that demonstrated unprecedented efficacy in this difficult-to-treat patient population. This review article outlines the main preclinical evaluations needed for CAR T cell product development.

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms.

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit-megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

Structural Determinants of Chimeric Antigen Receptor Design.

Chimeric antigen receptor (CAR) T cell therapy consists of the gene transfer of a cassette encoding a receptor capable of redirecting the transduced T cell toward a specific cytotoxic response against tumor cells. The therapy has been providing a new perspective on some hematologic malignancies, such as CD19+ lymphomas and acute lympho-blastic leukemia. CAR-T cell-based therapies are now approved for commercial distribution in different countries. Over the years, several modifications were necessary in the CAR structure to get it to its current results. CAR-T strategies still have plenty of room for improvement in order to improve clinical benefits and to overcome some of the limitations that still impair broader application. One main issue is the dysfunctional acquired phenotype, provoked by tumor inhibitory molecules or even exacerbated signaling by the CAR molecule itself. In this regard, Many research groups focus on discrete incremental modifications in each of the CAR molecule domains of the conventional structure looking for better response. Among these redesign strategies are the modulation of the binding affinity, use of costimulatory molecule ligands, and control of intracellular signaling. This review focuses on the newest reports covering structure changes in the CAR molecule capable of eliciting improved responses by transduced cells.

CAR-T cells leave the comfort zone: current and future applications beyond cancer.

Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in the immunotherapy field and has achieved great success following its approval in 2017 for the treatment of B cell malignancies. While CAR-T cells are mostly applied as anti-tumor therapy in the present, their initial concept was aimed at a more general purpose of targeting membrane antigens, thus translating in many potential applications. Since then, several studies have assessed the use of CAR-T cells toward non-malignant pathologies such as autoimmune diseases, infectious diseases and, more recently, cardiac fibrosis, and cellular senescence. In this review, we present the main findings and implications of CAR-based therapies for non-malignant conditions.

CRISPR / Cas-9 Vector System: Targets UL-39 and Inhibits HSV-1 Replication in Vitro

HSV-1 affects approximately 67% of the world population. Here, we sought to use the CRISPR / Cas9 system with the UL39 target, essential for virus replication. The sgRNA sequence was inserted into plasmid (PX459-UL39). Vero cells were transfected with PX459-UL39, and inhibition of viral replication was assessed 24 and 48 hours later using plaque assays and fluorescence and qPCR. Fluorescence analyzes revealed the presence of anti-HSV-1 CRISPR/Cas9 within Vero cells, and qPCR showed that the viral load decreased by> 95% of cells transfected with anti-HSV-1 CRISPR / Cas9. Our data demonstrate the usefulness of the PX459-UL39 to inhibit HSV-1 infection.

Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats.

Angiogenesis is considered to mediate the beneficial effects of mesenchymal cell therapy in spinal cord injury. After a moderate balloon-compression injury in rats, injections of either human adipose tissue-derived stromal/stem cells (hADSCs) or their conditioned culture media (CM-hADSC) elicited angiogenesis around the lesion site. Both therapies increased vascular density, but the presence of hADSCs in the tissue was required for the full maturation of new blood vessels. Only animals that received hADSC significantly improved their open field locomotion, assessed by the BBB score. Animals that received CM-hADSC only, presented haemorrhagic areas and lack pericytes. Proteomic analyses of human angiogenesis-related factors produced by hADSCs showed that both pro- and anti-angiogenic factors were produced by hADSCs in vitro, but only those related to vessel maturation were detectable in vivo. hADSCs produced PDGF-AA only after insertion into the injured spinal cord. hADSCs attracted resident pericytes expressing NG2, α-SMA, PDGF-Rß and nestin to the lesion, potentially contributing to blood vessel maturation. We conclude that the presence of hADSCs in the injured spinal cord is essential for tissue repair.

Senolytic chimeric antigen receptor (CAR) T cell: driving the immune system to fight cell senescence.

We discuss in this News and Commentary article the implications of senolytic chimeric antigen receptor T cells.