Gene therapy in pediatrics - Clinical studies and approved drugs (as of 2023).

Gene therapy in pediatrics represents a cutting-edge therapeutic strategy for treating a range of genetic disorders that manifest in childhood. Gene therapy involves the modification or correction of a mutated gene or the introduction of a functional gene into a patient's cells. In general, it is implemented through two main modalities namely ex vivo gene therapy and in vivo gene therapy. Currently, a noteworthy array of gene therapy products has received valid market authorization, with several others in various stages of the approval process. Additionally, a multitude of clinical trials are actively underway, underscoring the dynamic progress within this field. Pediatric genetic disorders in the fields of hematology, oncology, vision and hearing loss, immunodeficiencies, neurological, and metabolic disorders are areas for gene therapy interventions. This review provides a comprehensive overview of the evolution and current progress of gene therapy-based treatments in the clinic for pediatric patients. It navigates the historical milestones of gene therapies, currently approved gene therapy products by the U.S. Food and Drug Administration (FDA) and/or European Medicines Agency (EMA) for children, and the promising future for genetic disorders. By providing a thorough compilation of approved gene therapy drugs and published results of completed or ongoing clinical trials, this review serves as a guide for pediatric clinicians to get a quick overview of the situation of clinical studies and approved gene therapy products as of 2023.

Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

Free Standing Dry and Stable Nanoporous Polymer Films Made through Mechanical Deformation.

A new straight forward approach to create nanoporous polymer membranes with well defined average pore diameters is presented. The method is based on fast mechanical deformation of highly entangled polymer films at high temperatures and a subsequent quench far below the glass transition temperature Tg . The process is first designed generally by simulation and then verified for the example of polystyrene films. The methodology does not need any chemical processing, supporting substrate, or self assembly process and is solely based on polymer inherent entanglement effects. Pore diameters are of the order of ten polymer reptation tube diameters. The resulting membranes are stable over months at ambient conditions and display remarkable elastic properties.

A combination of metformin and epigallocatechin gallate potentiates glioma chemotherapy in vivo.

Glioma is the most devastating high-grade tumor of the central nervous system, with dismal prognosis. Existing treatment modality does not provide substantial benefit to patients and demands novel strategies. One of the first-line treatments for glioma, temozolomide, provides marginal benefit to glioma patients. Repurposing of existing non-cancer drugs to treat oncology patients is gaining momentum in recent years. In this study, we investigated the therapeutic benefits of combining three repurposed drugs, namely, metformin (anti-diabetic) and epigallocatechin gallate (green tea-derived antioxidant) together with temozolomide in a glioma-induced xenograft rat model. Our triple-drug combination therapy significantly inhibited tumor growth in vivo and increased the survival rate (50%) of rats when compared with individual or dual treatments. Molecular and cellular analyses revealed that our triple-drug cocktail treatment inhibited glioma tumor growth in rat model through ROS-mediated inactivation of PI3K/AKT/mTOR pathway, arrest of the cell cycle at G1 phase and induction of molecular mechanisms of caspases-dependent apoptosis.In addition, the docking analysis and quantum mechanics studies performed here hypothesize that the effect of triple-drug combination could have been attributed by their difference in molecular interactions, that maybe due to varying electrostatic potential. Thus, repurposing metformin and epigallocatechin gallate and concurrent administration with temozolomide would serve as a prospective therapy in glioma patients.

Haploidentical hematopoietic stem cell transplantation as individual treatment option in pediatric patients with very high-risk sarcomas.

Background: Prognosis of children with primary disseminated or metastatic relapsed sarcomas remains dismal despite intensification of conventional therapies including high-dose chemotherapy. Since haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is effective in the treatment of hematological malignancies by mediating a graft versus leukemia effect, we evaluated this approach in pediatric sarcomas as well. Methods: Patients with bone Ewing sarcoma or soft tissue sarcoma who received haplo-HSCT as part of clinical trials using CD3+ or TCRα/ß+ and CD19+ depletion respectively were evaluated regarding feasibility of treatment and survival. Results: We identified 15 patients with primary disseminated disease and 14 with metastatic relapse who were transplanted from a haploidentical donor to improve prognosis. Three-year event-free survival (EFS) was 18,1% and predominantly determined by disease relapse. Survival depended on response to pre-transplant therapy (3y-EFS of patients in complete or very good partial response: 36,4%). However, no patient with metastatic relapse could be rescued. Conclusion: Haplo-HSCT for consolidation after conventional therapy seems to be of interest for some, but not for the majority of patients with high-risk pediatric sarcomas. Evaluation of its future use as basis for subsequent humoral or cellular immunotherapies is necessary.

Generation of a heterozygous and a homozygous CSF1R knockout line from iPSC using CRISPR/Cas9.

Mutations in Colony-stimulating factor 1 receptor (CSF1R) lead to CSF1R-related leukoencephalopathy, also known as Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a rapidly progressing neurodegenerative disease with severe cognitive and motor impairment. In this study, a homozygous and a heterozygous CSF1R knockout induced pluripotent stem cell (iPSC) line were generated by CRISPR/Cas9-based gene editing. These in vitro models will provide a helpful tool for investigating the still largely unknown pathophysiology of CSF1R-related leukoencephalopathy.

Transferring measurable residual disease measurement in pediatric acute lymphoblastic leukemia from quantitative real-time PCR to digital droplet PCR.

BACKGROUND: Since the measurement of measurable residual disease (MRD) is part of clinical routine examination for children affected with acute lymphoblastic leukemia (ALL), continuous efforts are made to improve its method, applicability and accuracy. Whereas quantitative real-time polymerase chain reaction (qPCR) is considered as the gold standard for MRD detection and endowed with international guidelines for implementation and evaluation, these do not yet exist for digital droplet PCR (ddPCR). However, advantages are seen in droplet partitioning for MRD measurement to allow absolute quantification without depending on reference samples. METHODS: In this study, 17 MRD targets of nine patients with childhood B-ALL were analyzed with qPCR and ddPCR, respectively. All patients were assigned to high risk group and had hematopoietic stem cell transplantation and CD19 antibody therapy for relapse prevention. Starting with the sequences and guidelines of qPCR and optimizing the protocol for ddPCR, the MRD targets could also be measured precisely with this novel method, using the same primer and probe sets as for qPCR. RESULTS: The already established MRD protocol of qPCR could be transferred to ddPCR and all 17 MRD targets were measured in dilution series reaching comparable Limit of detection levels with both PCR methods. CONCLUSIONS: With a given qPCR protocol and some experience in conventional MRD monitoring, it is conceivable to transfer the procedure of MRD measurement to ddPCR technology. Our data is in line with other studies which are summarized and discussed here as well to facilitate the transfer of MRD diagnostics to ddPCR.

Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of ß-Hemoglobinopathies.

Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated clinical-scale production of genetically modified hematopoietic stem and progenitor cells (HSPCs) using the CliniMACS Prodigy® system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the BCL11A gene, clustered regularly interspaced short palindromic repeat (CRISPR) target in ongoing clinical trials for ß-thalassemia and sickle-cell disease treatment, was disrupted by the CRISPR-Cas9 system simulating a large-scale clinical scenario, yielding 100 million HSPCs with high editing efficiency. In vitro erythroid differentiation and high-performance liquid chromatography analyses corroborated fetal hemoglobin resurgence in edited samples, supporting the feasibility of running the complete process of HSPC gene editing in an automated closed system.

Corrigendum: A combination of metformin and epigallocatechin gallate potentiates glioma chemotherapy in vivo.

[This corrects the article DOI: 10.3389/fphar.2023.1096614.].

Challenges in Gene Therapy for Somatic Reverted Mosaicism in X-Linked Combined Immunodeficiency by CRISPR/Cas9 and Prime Editing.

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.

Preclinical Evaluation of CRISPR-Edited CAR-NK-92 Cells for Off-the-Shelf Treatment of AML and B-ALL.

Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines' cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.

Cohesion Gain Induced by Nanosilica Consolidants for Monumental Stone Restoration.

Mineral nanoparticle suspensions with consolidating properties have been successfully applied in the restoration of weathered architectural surfaces. However, the design of these consolidants is usually stone-specific and based on trial and error, which prevents their robust operation for a wide range of highly heterogeneous monumental stone materials. In this work, we develop a facile and versatile method to systematically study the consolidating mechanisms in action using a surface forces apparatus (SFA) with real-time force sensing and an X-ray surface forces apparatus (X-SFA). We directly assess the mechanical tensile strength of nanosilica-treated single mineral contacts and show a sharp increase in their cohesion. The smallest used nanoparticles provide an order of magnitude stronger contacts. We further resolve the microstructures and forces acting during evaporation-driven, capillary-force-induced nanoparticle aggregation processes, highlighting the importance of the interactions between the nanoparticles and the confining mineral walls. Our novel SFA-based approach offers insight into nano- and microscale mechanisms of consolidating silica treatments, and it can aid the design of nanomaterials used in stone consolidation.

Outstanding Charge Mobility by Band Transport in Two-Dimensional Semiconducting Covalent Organic Frameworks.

Two-dimensional covalent organic frameworks (2D COFs) represent a family of crystalline porous polymers with a long-range order and well-defined open nanochannels that hold great promise for electronics, catalysis, sensing, and energy storage. To date, the development of highly conductive 2D COFs has remained challenging due to the finite π-conjugation along the 2D lattice and charge localization at grain boundaries. Furthermore, the charge transport mechanism within the crystalline framework remains elusive. Here, time- and frequency-resolved terahertz spectroscopy reveals intrinsically Drude-type band transport of charge carriers in semiconducting 2D COF thin films condensed by 1,3,5-tris(4-aminophenyl)benzene (TPB) and 1,3,5-triformylbenzene (TFB). The TPB-TFB COF thin films demonstrate high photoconductivity with a long charge scattering time exceeding 70 fs at room temperature which resembles crystalline inorganic materials. This corresponds to a record charge carrier mobility of 165 ± 10 cm2 V-1 s-1, vastly outperforming that of the state-of-the-art conductive COFs. These results reveal TPB-TFB COF thin films as promising candidates for organic electronics and catalysis and provide insights into the rational design of highly crystalline porous materials for efficient and long-range charge transport.

Time-dependent analysis of adoptive immunotherapy following sequential FLAMSA-reduced intensity conditioning and allogeneic hematopoietic stem cell transplantation in patients with high-risk myeloid neoplasia.

Prophylactic donor lymphocyte infusions (DLI) are part of the sequential FLAMSA-reduced intensity conditioning (RIC) regimen to cure high risk myeloid neoplasia with allogeneic hematopoietic stem cell transplantation (HSCT). Although DLI themselves carry significant risks, their prophylactic use has not been analyzed in a time-dependent manner. One hundred and fourteen patients underwent FLAMSA-RIC HSCT between 2013 and 2020. Next to Kaplan-Meier estimation of overall, disease-free, and graft-versus-host relapse-free survival (OS, DFS, GRFS), cumulative incidences of relapse and death in remission were calculated in a competing risk model. Additionally, the contribution of prophylactic and preemptive DLI as time-dependent covariates was assessed using a time-varying model toward DFS (Simon-Makuch method, Mantel-Byar test). At 2 years, OS was 45.2% [95% CI 36.7-55.7%], DFS 31.8% [95% CI 24-42.2%] and GRFS 11.3 [95% CI 6.5-19.8]. Neither prophylactic nor preemptive DLI showed a significant influence on DFS when considered time-dependent covariates (Mantel-Byar, p = .3). This was further corroborated in competing risk analysis with DLI as time-dependent covariates. Both prophylactic and preemptive DLI miss significance in their impact on survival within a high-risk cohort in a time-varying model. Controlled trials to address the impact of postgrafting immunotherapy approaches are needed.

A Mutation-Agnostic Hematopoietic Stem Cell Gene Therapy for Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by mutations in the Arylsulfatase-A (ARSA) gene. The enzyme plays a key role in sulfatide metabolism in brain cells, and its deficiency leads to neurodegeneration. The clinical manifestations of MLD include stagnation and decline of motor and cognitive function, leading to premature death with limited standard treatment options. Here, we describe a mutation-agnostic hematopoietic stem and progenitor cell (HSPC) gene therapy using CRISPR-Cas9 and AAV6 repair template as a prospective treatment option for MLD. Our strategy achieved efficient insertions and deletions (>87%) and a high level of gene integration (>47%) at the ARSA locus in human bone marrow-derived HSPCs, with no detectable off-target editing. As a proof of concept, we tested our mutation-agnostic therapy in HSPCs derived from two MLD patients with distinct mutations and demonstrated restoration of ARSA enzyme activity (>30-fold improvement) equivalent to healthy adults. In summary, our investigation enabled a mutation-agnostic therapy for MLD patients with proven efficacy and strong potential for clinical translation.

CRISPR medicine for blood disorders: Progress and challenges in delivery.

Blood disorders are a group of diseases including hematological neoplasms, clotting disorders and orphan immune deficiency diseases that affects human health. Current improvements in genome editing based therapeutics demonstrated preclinical and clinical proof to treat different blood disorders. Genome editing components such as Cas nucleases, guide RNAs and base editors are supplied in the form of either a plasmid, an mRNA, or a ribonucleoprotein complex. The most common delivery vehicles for such components include viral vectors (e.g., AAVs and RV), non-viral vectors (e.g., LNPs and polymers) and physical delivery methods (e.g., electroporation and microinjection). Each of the delivery vehicles specified above has its own advantages and disadvantages and the development of a safe transferring method for ex vivo and in vivo application of genome editing components is still a big challenge. Moreover, the delivery of genome editing payload to the target blood cells possess key challenges to provide a possible cure for patients with inherited monogenic blood diseases and hematological neoplastic tumors. Here, we critically review and summarize the progress and challenges related to the delivery of genome editing elements to relevant blood cells in an ex vivo or in vivo setting. In addition, we have attempted to provide a future clinical perspective of genome editing to treat blood disorders with possible clinical grade improvements in delivery methods.

Immunomonitoring of Stage IV Relapsed Neuroblastoma Patients Undergoing Haploidentical Hematopoietic Stem Cell Transplantation and Subsequent GD2 (ch14.18/CHO) Antibody Treatment.

Haploidentical stem cell transplantation (haplo SCT) in Stage IV neuroblastoma relapsed patients has been proven efficacious, while immunotherapy utilizing the anti-GD2 antibody dinutuximab beta has become a standard treatment for neuroblastoma. The combinatorial therapy of haplo SCT and dinutuximab may potentiate the efficacy of the immunotherapy. To gain further understanding of the synergistic effects, functional immunomonitoring was assessed during the clinical trial CH14.18 1021 Antibody and IL2 After haplo SCT in Children with Relapsed Neuroblastoma (NCT02258815). Rapid immune reconstitution of the lymphoid compartment was confirmed, with clinically relevant dinutuximab serum levels found in all patients over the course of treatment. Only one patient developed human anti-chimeric antibodies (HACAs). In-patient monitoring revealed highly functional NK cell posttransplant capable of antibody-dependent cellular cytotoxicity (ADCC). Degranulation of NK cell subsets revealed a significant response increased by dinutuximab. This was irrespective of the KIR receptor-ligand constellation within the NK subsets, defined by the major KIR receptors CD158a, CD158b, and CD158e. Moreover, complement-dependent cytotoxicity (CDC) was shown to be an extremely potent effector-cell independent mechanism of tumor cell lysis, with a clear positive correlation to GD2 expression on the cancer cells as well as to the dinutuximab concentrations. The ex vivo testing of patient-derived effector cells and the sera collected during dinutuximab therapy demonstrated both high functionality of the newly established lymphoid immune compartment and provided confidence that the antibody dosing regimen was sufficient over the duration of the dinutuximab therapy (up to nine cycles in a 9-month period). During the course of the dinutuximab therapy, proinflammatory cytokines and markers (sIL2R, TNFa, IL6, and C reactive protein) were significantly elevated indicating a strong anti-GD2 immune response. No impact of FcGR polymorphism on event-free and overall survival was found. Collectively, this study has shown that in-patient functional immunomonitoring is feasible and valuable in contributing to the understanding of anti-cancer combinatorial treatments such as haplo SCT and antibody immunotherapy.

In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment.

BACKGROUND: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). METHODS: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. RESULTS: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. CONCLUSIONS: The strong cytotoxic effect of a CD276-specific CAR in combination with an "off-the-shelf" NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.

Universal Gene Correction Approaches for ß-hemoglobinopathies Using CRISPR-Cas9 and Adeno-Associated Virus Serotype 6 Donor Templates.

Mutations in the human ß-globin gene are the cause of ß-hemoglobinopathies, one of the most common inherited single-gene blood disorders in the world. Novel therapeutic approaches are based on lentiviral vectors (LVs) or CRISPR-Cas9-mediated gene disruption to express adult hemoglobin (HbA), or to reactivate the completely functional fetal hemoglobin, respectively. Nonetheless, LVs present a risk of insertional mutagenesis, while gene-disrupting transcription factors (BCL11A, KLF1) involved in the fetal-to-adult hemoglobin switch might generate dysregulation of other cellular processes. Therefore, universal gene addition/correction approaches combining CRISPR-Cas9 and homology directed repair (HDR) by delivering a DNA repair template through adeno-associated virus could mitigate the limitations of both lentiviral gene transfer and gene disruption strategies, ensuring targeted integration and controlled transgene expression. In this study, we attained high rates of gene addition (up to 12%) and gene correction (up to 38%) in hematopoietic stem and progenitor cells from healthy donors without any cell sorting/enrichment or the application of HDR enhancers. Furthermore, these approaches were tested in heterozygous (ß0/ß+) and homozygous (ß0/ß0, ß+/ß+) ß-thalassemia patients, achieving a significant increase in HbA and demonstrating the universal therapeutic potential of this study for the treatment of ß-hemoglobinopathies.