Correction of Griscelli Syndrome Type 2 causing mutations in the gene with CRISPR/Cas9.

Background/aim: Griscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the gene. The current treatment consists of hematopoietic stem cell transplantation, but a lack of suitable donors warrants the development of alternative treatment strategies, including gene therapy. The development of mutation-specific clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing technology has opened the way for custom-designed gene correction of patient-derived stem cells.

Assessment of non-myelotoxic agents as a preparatory regimen for hematopoietic stem cell gene therapy.

RAG2 deficiency is characterized by a lack of B and T lymphocytes, causing severe lethal infections. Currently, RAG2 deficiency is treated with a Hematopoietic Stem Cell transplantation (HSCT). Most conditioning regimens used before HSCT consist of alkylating myelotoxic agents with or without irradiation and affect growth and development of pediatric patients.

Human post-mortem organotypic brain slice cultures: a tool to study pathomechanisms and test therapies.

Human brain experimental models recapitulating age- and disease-related characteristics are lacking. There is urgent need for human-specific tools that model the complex molecular and cellular interplay between different cell types to assess underlying disease mechanisms and test therapies. Here we present an adapted ex vivo organotypic slice culture method using human post-mortem brain tissue cultured at an air-liquid interface to also study brain white matter.

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML.

Background: Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft.

In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models.

Lentiviral gene therapy with IGF2-tagged GAA normalizes the skeletal muscle proteome in murine Pompe disease.

Pompe disease is a lysosomal storage disorder caused by deficiency of acid alpha-glucosidase (GAA), resulting in glycogen accumulation with profound pathology in skeletal muscle. We recently developed an optimized form of lentiviral gene therapy for Pompe disease in which a codon-optimized version of the GAA transgene (LV-GAAco) was fused to an insulin-like growth factor 2 (IGF2) peptide (LV-IGF2.GAAco), to promote cellular uptake via the cation-independent mannose-6-phosphate/IGF2 receptor.

Gene and Cellular Therapies for Leukodystrophies.

Leukodystrophies are a heterogenous group of inherited, degenerative encephalopathies, that if left untreated, are often lethal at an early age. Although some of the leukodystrophies can be treated with allogeneic hematopoietic stem cell transplantation, not all patients have suitable donors, and new treatment strategies, such as gene therapy, are rapidly being developed. Recent developments in the field of gene therapy for severe combined immune deficiencies, Leber's amaurosis, epidermolysis bullosa, Duchenne's muscular dystrophy and spinal muscular atrophy, have paved the way for the treatment of leukodystrophies, revealing some of the pitfalls, but overall showing promising results.

Focal lesions following intracerebral gene therapy for mucopolysaccharidosis IIIA.

Objective: Mucopolysaccharidosis type IIIA (MPSIIIA) caused by recessive SGSH variants results in sulfamidase deficiency, leading to neurocognitive decline and death. No disease-modifying therapy is available. The AAVance gene therapy trial investigates AAVrh.

Screening chimeric GAA variants in preclinical study results in hematopoietic stem cell gene therapy candidate vectors for Pompe disease.

Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations.

IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy.

Pompe disease is caused by deficiency of acid α-glucosidase (GAA), resulting in glycogen accumulation in various tissues, including cardiac and skeletal muscles and the central nervous system (CNS). Enzyme replacement therapy (ERT) improves cardiac, motor, and respiratory functions but is limited by poor cellular uptake and its inability to cross the blood-brain barrier. Previously, we showed that hematopoietic stem cell (HSPC)-mediated lentiviral gene therapy (LVGT) with codon-optimized (LV-) caused glycogen reduction in heart, skeletal muscles, and partially in the brain at high vector copy number (VCN).

Lentiviral gene therapy prevents anti-human acid α-glucosidase antibody formation in murine Pompe disease.

Enzyme replacement therapy (ERT) is the current standard treatment for Pompe disease, a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). ERT has shown to be lifesaving in patients with classic infantile Pompe disease. However, a major drawback is the development of neutralizing antibodies against ERT.

Gene Therapy Developments for Pompe Disease.

Pompe disease is an inherited neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The most severe form is infantile-onset Pompe disease, presenting shortly after birth with symptoms of cardiomyopathy, respiratory failure and skeletal muscle weakness. Late-onset Pompe disease is characterized by a slower disease progression, primarily affecting skeletal muscles.

Efficient lentiviral transduction method to gene modify cord blood CD8 T cells for cancer therapy applications.

Adoptive T cell therapy utilizing tumor-specific autologous T cells has shown promising results for cancer treatment. However, the limited numbers of autologous tumor-associated antigen (TAA)-specific T cells and the functional aberrancies, due to disease progression or treatment, remain factors that may significantly limit the success of the therapy. The use of allogeneic T cells, such as umbilical cord blood (CB) derived, overcomes these issues but requires gene modification to induce a robust and specific anti-tumor effect.

Gene Augmentation and Editing to Improve TCR Engineered T Cell Therapy against Solid Tumors.

Recent developments in gene engineering technologies have drastically improved the therapeutic treatment options for cancer patients. The use of effective chimeric antigen receptor T (CAR-T) cells and recombinant T cell receptor engineered T (rTCR-T) cells has entered the clinic for treatment of hematological malignancies with promising results. However, further fine-tuning, to improve functionality and safety, is necessary to apply these strategies for the treatment of solid tumors.

Lentiviral Hematopoietic Stem Cell Gene Therapy Corrects Murine Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years.

A "No-Touch" Antibody-Staining Method of Adherent Cells for High-Throughput Flow Cytometry in 384-Well Microplate Format for Cell-Based Drug Library Screening.

In the last decade, screening compound libraries on live cells has become an important step in drug discovery. The abundance of compounds in these libraries requires effective high-throughput (HT) analyzing methods. Although current cell-based assay protocols are suitable for HT analyses, the analysis itself is often restrained to simple, singular outcomes.

Transplantation, gene therapy and intestinal pathology in MNGIE patients and mice.

Background: Gastrointestinal complications are the main cause of death in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Available treatments often restore biochemical homeostasis, but fail to cure gastrointestinal symptoms.

Methods: We evaluated the small intestine neuromuscular pathology of an untreated MNGIE patient and two recipients of hematopoietic stem cells, focusing on enteric neurons and glia.

Strategies to Genetically Modulate Dendritic Cells to Potentiate Anti-Tumor Responses in Hematologic Malignancies.

Dendritic cell (DC) vaccination has been investigated as a potential strategy to target hematologic malignancies, while generating sustained immunological responses to control potential future relapse. Nonetheless, few clinical trials have shown robust long-term efficacy. It has been suggested that a combination of surmountable shortcomings, such as selection of utilized DC subsets, DC loading and maturation strategies, as well as tumor-induced immunosuppression may be targeted to maximize anti-tumor responses of DC vaccines.

Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks.

Use of cord blood derived T-cells in cancer immunotherapy: milestones achieved and future perspectives.

Hematopoietic cell transplantation is a potentially lifesaving procedure for patients with hematological malignancies who are refractory to conventional chemotherapy and/or irradiation treatment. Umbilical cord blood (CB) transplantation, as a hematopoietic stem and progenitor cell (HSPC) source, has several advantages over bone marrow transplantation with respect to matching and prompt availability for transplantation. Additionally, CB has some inherent features, such as rapid expansion of T cells, lower prevalence of graft-versus-host disease and higher graft versus tumor efficacy that make this HSPC cell source more favorable over other HSPC sources.

Efficacy of lentivirus-mediated gene therapy in an Omenn syndrome recombination-activating gene 2 mouse model is not hindered by inflammation and immune dysregulation.

Background: Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene (RAG) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor.

Enhancement of mouse hematopoietic stem/progenitor cell function via transient gene delivery using integration-deficient lentiviral vectors.

Integration-deficient lentiviruses (IdLVs) deliver genes effectively to tissues but are lost rapidly from dividing cells. This property can be harnessed to express transgenes transiently to manipulate cell biology. Here, we demonstrate the utility of short-term gene expression to improve functional potency of hematopoietic stem and progenitor cells (HSPCs) during transplantation by delivering HOXB4 and Angptl3 using IdLVs to enhance the engraftment of HSPCs.