Hematopoietic stem cell gene editing rescues B-cell development in X-linked agammaglobulinemia.

BACKGROUND: X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA. METHODS: To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured in vitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output in vivo. RESULTS: A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery. CONCLUSIONS: Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.

Investigating adverse genomic and regulatory changes caused by replacement of the full-length CFTR cDNA using Cas9 and AAV.

A "universal strategy" replacing the full-length CFTR cDNA may treat >99% of people with cystic fibrosis (pwCF), regardless of their specific mutations. Cas9-based gene editing was used to insert the CFTR cDNA and a truncated CD19 (tCD19) enrichment tag at the CFTR locus in airway basal stem cells. This strategy restores CFTR function to non-CF levels. Here, we investigate the safety of this approach by assessing genomic and regulatory changes after CFTR cDNA insertion. Safety was first assessed by quantifying genetic rearrangements using CAST-seq. After validating restored CFTR function in edited and enriched airway cells, the CFTR locus open chromatin profile was characterized using ATAC-seq. The regenerative potential and differential gene expression in edited cells was assessed using scRNA-seq. CAST-seq revealed a translocation in ∼0.01% of alleles primarily occurring at a nononcogenic off-target site and large indels in 1% of alleles. The open chromatin profile of differentiated airway epithelial cells showed no appreciable changes, except in the region corresponding to the CFTR cDNA and tCD19 cassette, indicating no detectable changes in gene regulation. Edited stem cells produced the same types of airway cells as controls with minimal alternations in gene expression. Overall, the universal strategy showed minor undesirable genomic changes.

CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.

Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Preclinical model for phenotypic correction of dystrophic epidermolysis bullosa by in vivo CRISPR-Cas9 delivery using adenoviral vectors.

Recessive dystrophic epidermolysis bullosa, a devastating skin fragility disease characterized by recurrent skin blistering, scarring, and a high risk of developing squamous cell carcinoma is caused by mutations in COL7A1, the gene encoding type VII collagen, which is the major component of the anchoring fibrils that bind the dermis and epidermis. Ex vivo correction of COL7A1 by gene editing in patients' cells has been achieved before. However, in vivo editing approaches are necessary to address the direct treatment of the blistering lesions characteristic of this disease. We have now generated adenoviral vectors for CRISPR-Cas9 delivery to remove exon 80 of COL7A1, which contains a highly prevalent frameshift mutation in Spanish patients. For in vivo testing, a humanized skin mouse model was used. Efficient viral transduction of skin was observed after excisional wounds generated with a surgical punch on regenerated patient skin grafts were filled with the adenoviral vectors embedded in a fibrin gel. Type VII collagen deposition in the basement membrane zone of the wounded areas treated with the vectors correlated with restoration of dermal-epidermal adhesion, demonstrating that recessive dystrophic epidermolysis bullosa (RDEB) patient skin lesions can be directly treated by CRISPR-Cas9 delivery in vivo.

Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq.

Genome editing has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by off-target effects of programmable nucleases. Here we describe chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq), a preclinical assay to identify and quantify chromosomal aberrations derived from on-target and off-target activities of CRISPR-Cas nucleases or transcriptional activator-like effector nucleases (TALENs), respectively, in human hematopoietic stem cells (HSCs). Depending on the employed designer nuclease, CAST-Seq detected translocations in 0%-0.5% of gene-edited human CD34+ HSCs, and up to 20% of on-target loci harbored gross rearrangements. Moreover, CAST-Seq detected distinct types of chromosomal aberrations, such as homology-mediated translocations, that are mediated by homologous recombination and not off-target activity. CAST-Seq is a sensitive assay able to identify and quantify unintended chromosomal rearrangements in addition to the more typical mutations at off-target sites. CAST-Seq analyses may be particularly relevant for therapeutic genome editing to enable thorough risk assessment before clinical application of gene-edited products.

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.

Correction: Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells.

[This corrects the article DOI: 10.1371/journal.pone.0112712.].

Gene Editing and Genotoxicity: Targeting the Off-Targets.

Gene editing technologies show great promise for application to human disease as a result of rapid developments in targeting tools notably based on ZFN, TALEN, and CRISPR-Cas systems. Precise modification of a DNA sequence is now possible in mature human somatic cells including stem and progenitor cells with increasing degrees of efficiency. At the same time new technologies are required to evaluate their safety and genotoxicity before widespread clinical application can be confidently implemented. A number of methodologies have now been developed in an attempt to predict expected and unexpected modifications occurring during gene editing. This review surveys the techniques currently available as state of the art, highlighting benefits and limitations, and discusses approaches that may achieve sufficient accuracy and predictability for application in clinical settings.

Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina.

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.

Genome and Epigenome Editing to Treat Disorders of the Hematopoietic System.

The possibility of editing complex genomes in a targeted fashion has revolutionized basic research as well as biomedical and biotechnological applications in the last 5 years. The targeted introduction of genetic changes has allowed researchers to create smart model systems for basic research, bio-engineers to modify crops and farm animals, and translational scientists to develop novel treatment approaches for inherited and acquired disorders for which curative treatment options are not yet available. With the rapid development of genome editing tools, in particular zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system, a wide range of therapeutic options have been-and will be-developed at an unprecedented speed, which will change the clinical routine of various disciplines in a revolutionary way. This review summarizes the fundamentals of genome editing and the current state of research. It particularly focuses on the advances made in employing engineered nucleases in hematopoietic stem cells for the treatment of primary immunodeficiencies and hemoglobinopathies, provides a perspective of combining gene editing with the chimeric antigen receptor T cell technology, and concludes by presenting targeted epigenome editing as a novel potential treatment option.

Correction of Recessive Dystrophic Epidermolysis Bullosa by Transposon-Mediated Integration of COL7A1 in Transplantable Patient-Derived Primary Keratinocytes.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects in type-VII collagen (C7), a protein encoded by the COL7A1 gene and essential for anchoring fibril formation at the dermal-epidermal junction. Gene therapy of RDEB is based on transplantation of autologous epidermal grafts generated from gene-corrected keratinocytes sustaining C7 deposition at the dermal-epidermal junction. Transfer of the COL7A1 gene is complicated by its very large size and repetitive sequence. This article reports a gene delivery approach based on the Sleeping beauty transposon, which allows integration of a full-length COL7A1 cDNA and secretion of C7 at physiological levels in RDEB keratinocytes without rearrangements or detrimental effects on their clonogenic potential. Skin equivalents derived from gene-corrected RDEB keratinocytes were tested in a validated preclinical model of xenotransplantation on immunodeficient mice, where they showed normal deposition of C7 at the dermal-epidermal junction and restoration of skin adhesion properties. These results indicate the feasibility and efficacy of a transposon-based gene therapy approach to RDEB.

Dominant and recessive mutations in rhodopsin activate different cell death pathways.

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.

Genomic analysis of Sleeping Beauty transposon integration in human somatic cells.

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity "sandwich" (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy applications.