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.

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.

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.

Depletion of Akt1 and Akt2 Impairs the Repair of Radiation-Induced DNA Double Strand Breaks via Homologous Recombination.

Homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and alternative NHEJ are major pathways that are utilized by cells for processing DNA double strand breaks (DNA-DSBs); their function plays an important role in the radiation resistance of tumor cells. Conflicting data exist regarding the role of Akt in homologous recombination (HR), i.e., the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT-knock-out and siRNA-mediated AKT-knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly demonstrated that HCT116 AKT1-KO and AKT2-KO cells have a significantly reduced Rad51 foci formation 6 h post irradiation versus parental cells. Depletion of Akt1 and Akt2 protein levels as well as inhibition of Akt kinase activity resulted in an increased number of residual-γH2AX in CENP-F positive cells mainly representing the S and G2 phase cells. Furthermore, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in stronger radiosensitivity of AKT1 and AKT2 knockout cells versus wild type cells. These data collectively show that both Akt1 and Akt2 are involved in DSBs repair through HRR.