Design and validation of a GMP stem cell manufacturing protocol for MPSII hematopoietic stem cell gene therapy.

Hematopoietic stem cell gene therapy (HSCGT) is a promising therapeutic strategy for the treatment of neurodegenerative, metabolic disorders. The approach involves the ex vivo introduction of a missing gene into patients' own stem cells via lentiviral-mediated transduction (TD). Once transplanted back into a fully conditioned patient, these genetically modified HSCs can repopulate the blood system and produce the functional protein, previously absent or non-functional in the patient, which can then cross-correct other affected cells in somatic organs and the central nervous system. We previously developed an HSCGT approach for the treatment of Mucopolysaccharidosis type II (MPSII) (Hunter syndrome), a debilitating pediatric lysosomal disorder caused by mutations in the iduronate-2-sulphatase (IDS) gene, leading to the accumulation of heparan and dermatan sulfate, which causes severe neurodegeneration, skeletal abnormalities, and cardiorespiratory disease. In HSCGT proof-of-concept studies using lentiviral IDS fused to a brain-targeting peptide ApoEII (IDS.ApoEII), we were able to normalize brain pathology and behavior of MPSII mice. Here we present an optimized and validated good manufacturing practice hematopoietic stem cell TD protocol for MPSII in preparation for first-in-man studies. Inclusion of TEs LentiBOOST and protamine sulfate significantly improved TD efficiency by at least 3-fold without causing adverse toxicity, thereby reducing vector quantity required.

Interleukin-1 blockade in patients with Wiskott-Aldrich Syndrome: a retrospective multinational case series.

Up to 70% of patients with Wiskott-Aldrich Syndrome (WAS) develop autoimmune and inflammatory manifestations. Dysregulation of interleukin (IL)-1 may be involved in their pathogenesis, yet there is little evidence on treatment with anti-IL-1 agents in these patients. We conducted a multicenter retrospective analysis of nine patients with WAS treated with anti-IL-1 agents (anakinra or canakinumab). All patients had prominent inflammatory manifestations, including systemic, cutaneous, articular, and intestinal symptoms; three patients presented with a severe systemic inflammatory syndrome since the first months of life. Corticosteroid therapy was associated with partial or no response, while treatment with anakinra or canakinumab resulted in prompt, often dramatic, responses in all patients, allowing bridging to gene therapy (four patients) or hematopoietic stem cell transplantation (HSCT, five patients). Treatment was overall well tolerated. Low donor myeloid chimerism developed in four patients after HSCT and was associated with the appearance or the recurrence of inflammatory manifestations. A second HSCT was performed in two patients, achieving full-donor chimerism and resolution of inflammatory manifestation, while the other two patients were treated with prolonged therapy with anti-IL-1 agents. Our experience demonstrates that some inflammatory manifestations of WAS are dependent on IL-1 and respond very well to its pharmacologic blockade.

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.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant.

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. In vitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

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.

The British Society for Gene and Cell Therapy at 20 (2003-2023).

The year 2023 marks the 20th anniversary of the British Society for Gene and Cell Therapy (BSGCT). In these 20 years, the field of gene and cell therapy has gone from promising strategy to clinical reality. This report describes the history, objectives, organization, and activities of BSGCT to advance research and practice of gene and cell therapy in the United Kingdom.

Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67phox-Deficient Chronic Granulomatous Disease.

Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34+ hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34+ cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34+ cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67phox-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67phox expression and functional correction of CD34+-derived neutrophils from a CGD patient.

Correction: The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0015730.].

AAV capsid bioengineering in primary human retina models.

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors.

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells.

Gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR-Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat hematological disorders, and clinical trials using these tools are now under way. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and their in vitro expansion for clinical application. In this study, we assessed the effect of different culture medium compositions on the ability of HSPCs to proliferate and undergo homology-directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex vivo culture. We demonstrated that by supplementing the culture medium with stem cell agonists and by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion. Overall, the implementation of this optimized ex vivo HSPC culture protocol can improve the efficacy, feasibility, and applicability of gene editing as a key step to unlocking the full therapeutic potential of this powerful technology.

Severe hematopoietic stem cell inflammation compromises chronic granulomatous disease gene therapy.

X-linked chronic granulomatous disease (CGD) is associated with defective phagocytosis, life-threatening infections, and inflammatory complications. We performed a clinical trial of lentivirus-based gene therapy in four patients (NCT02757911). Two patients show stable engraftment and clinical benefits, whereas the other two have progressively lost gene-corrected cells. Single-cell transcriptomic analysis reveals a significantly lower frequency of hematopoietic stem cells (HSCs) in CGD patients, especially in the two patients with defective engraftment. These two present a profound change in HSC status, a high interferon score, and elevated myeloid progenitor frequency. We use elastic-net logistic regression to identify a set of 51 interferon genes and transcription factors that predict the failure of HSC engraftment. In one patient, an aberrant HSC state with elevated CEBPß expression drives HSC exhaustion, as demonstrated by low repopulation in a xenotransplantation model. Targeted treatments to protect HSCs, coupled to targeted gene expression screening, might improve clinical outcomes in CGD.

Replication competent retrovirus testing (RCR) in the National Gene Vector Biorepository: No evidence of RCR in 1,595 post-treatment peripheral blood samples obtained from 60 clinical trials.

The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.

Lentiviral Gene Transfer Corrects Immune Abnormalities in XIAP Deficiency.

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) deficiency is a severe immunodeficiency with clinical features including hemophagocytic lymphohistiocytosis (HLH) and inflammatory bowel disease (IBD) due to defective NOD2 responses. Management includes immunomodulatory therapies and hematopoietic stem cell transplant (HSCT). However, this cohort is particularly susceptible to the chemotherapeutic regimens and acutely affected by graft-vs-host disease (GvHD), driving poor long-term survival in transplanted patients. Autologous HSC gene therapy could offer an alternative treatment option and would abrogate the risks of alloreactivity. METHODS: Hematopoietic progenitor (Lin-ve) cells from XIAPy/- mice were transduced with a lentiviral vector encoding human XIAP cDNA before transplantation into irradiated XIAP y/- recipients. After 12 weeks animals were challenged with the dectin-1 ligand curdlan and recovery of innate immune function was evaluated though analysis of inflammatory cytokines, body weight, and splenomegaly. XIAP patient-derived CD14+ monocytes were transduced with the same vector and functional recovery was demonstrated using in vitro L18-MDP/NOD2 assays. RESULTS: In treated XIAPy/- mice, ~40% engraftment of gene-corrected Lin-ve cells led to significant recovery of weight loss, splenomegaly, and inflammatory cytokine responses to curdlan, comparable to wild-type mice. Serum IL-6, IL-10, MCP-1, and TNF were significantly reduced 2-h post-curdlan administration in non-corrected XIAPy/- mice compared to wild-type and gene-corrected animals. Appropriate reduction of inflammatory responses was observed in gene-corrected mice, whereas non-corrected mice developed an inflammatory profile 9 days post-curdlan challenge. In gene-corrected patient CD14+ monocytes, TNF responses were restored following NOD2 activation with L18-MDP. CONCLUSION: Gene correction of HSCs recovers XIAP-dependent immune defects and could offer a treatment option for patients with XIAP deficiency.

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.

Partial human Janus kinase 1 deficiency predominantly impairs responses to interferon gamma and intracellular control of mycobacteria.

Purpose: Janus kinase-1 (JAK1) tyrosine kinase mediates signaling from multiple cytokine receptors, including interferon alpha/beta and gamma (IFN-α/ß and IFN-γ), which are important for viral and mycobacterial protection respectively. We previously reported autosomal recessive (AR) hypomorphic JAK1 mutations in a patient with recurrent atypical mycobacterial infections and relatively minor viral infections. This study tests the impact of partial JAK1 deficiency on cellular responses to IFNs and pathogen control. Methods: We investigated the role of partial JAK1 deficiency using patient cells and cell models generated with lentiviral vectors expressing shRNA. Results: Partial JAK1 deficiency impairs IFN-γ-dependent responses in multiple cell types including THP-1 macrophages, Epstein-Barr Virus (EBV)-transformed B cells and primary dermal fibroblasts. In THP-1 myeloid cells, partial JAK1 deficiency reduced phagosome acidification and apoptosis and resulted in defective control of mycobacterial infection with enhanced intracellular survival. Although both EBV-B cells and primary dermal fibroblasts with partial JAK1 deficiency demonstrate reduced IFN-α responses, control of viral infection was impaired only in patient EBV-B cells and surprisingly intact in patient primary dermal fibroblasts. Conclusion: Our data suggests that partial JAK1 deficiency predominantly affects susceptibility to mycobacterial infection through impact on the IFN-γ responsive pathway in myeloid cells. Susceptibility to viral infections as a result of reduced IFN-α responses is variable depending on cell type. Description of additional patients with inherited JAK1 deficiency will further clarify the spectrum of bacterial and viral susceptibility in this condition. Our results have broader relevance for anticipating infectious complications from the increasing use of selective JAK1 inhibitors.

Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a central regulator of immunity. TRAF3 is often somatically mutated in B cell malignancies, but its role in human immunity is not defined. Here, in five unrelated families, we describe an immune dysregulation syndrome of recurrent bacterial infections, autoimmunity, systemic inflammation, B cell lymphoproliferation, and hypergammaglobulinemia. Affected individuals each had monoallelic mutations in TRAF3 that reduced TRAF3 expression. Immunophenotyping showed that patients' B cells were dysregulated, exhibiting increased nuclear factor-κB 2 activation, elevated mitochondrial respiration, and heightened inflammatory responses. Patients had mild CD4+ T cell lymphopenia, with a reduced proportion of naïve T cells but increased regulatory T cells and circulating T follicular helper cells. Guided by this clinical phenotype, targeted analyses demonstrated that common genetic variants, which also reduce TRAF3 expression, are associated with an increased risk of B cell malignancies, systemic lupus erythematosus, higher immunoglobulin levels, and bacterial infections in the wider population. Reduced TRAF3 conveys disease risks by driving B cell hyperactivity via intrinsic activation of multiple intracellular proinflammatory pathways and increased mitochondrial respiration, with a likely contribution from dysregulated T cell help. Thus, we define monogenic TRAF3 haploinsufficiency syndrome and demonstrate how common TRAF3 variants affect a range of human diseases.