Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However, responses triggered by programmable nucleases in HSPCs are poorly ...characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here, we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction, cell-cycle progression, and transcriptional responses in HSPC subpopulations, with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation, constraining proliferation, yield, and engraftment of edited HSPCs. However, functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs.
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•DNA DSBs induced by programmable nucleases transiently activate the DDR in HSPCs•Single-cell transcriptomics show that induced DSBs activate the p53 pathway•AAV6-mediated genome editing aggravates p53 activation and delays HSPC proliferation•Transient p53 inhibition alleviates clonogenic and repopulation defects in edited HSPCs
Precise gene editing has the potential to treat immune and hematological diseases. Genovese, Naldini, Di Micco, and colleagues now show that gene-editing procedures are well tolerated by hematopoietic stem cells and provide molecular evidence of the feasibility of seamless gene editing, strengthening translation of such approaches to humans.
Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die ...within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.
Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant MPSIH). However, this treatment is only partially curative ...and is associated with complications.
We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years.
We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts.
The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).
Summary Background Metachromatic leukodystrophy (a deficiency of arylsulfatase A ARSA) is a fatal demyelinating lysosomal disease with no approved treatment. We aimed to assess the long-term outcomes ...in a cohort of patients with early-onset metachromatic leukodystrophy who underwent haemopoietic stem-cell gene therapy (HSC-GT). Methods This is an ad-hoc analysis of data from an ongoing, non-randomised, open-label, single-arm phase 1/2 trial, in which we enrolled patients with a molecular and biochemical diagnosis of metachromatic leukodystrophy (presymptomatic late-infantile or early-juvenile disease or early-symptomatic early-juvenile disease) at the Paediatric Clinical Research Unit, Ospedale San Raffaele, in Milan. Trial participants received HSC-GT, which consisted of the infusion of autologous HSCs transduced with a lentiviral vector encoding ARSA cDNA, after exposure-targeted busulfan conditioning. The primary endpoints of the trial are safety (toxicity, absence of engraftment failure or delayed haematological reconstitution, and safety of lentiviral vector-tranduced cell infusion) and efficacy (improvement in Gross Motor Function Measure GMFM score relative to untreated historical controls, and ARSA activity, 24 months post-treatment) of HSC-GT. For this ad-hoc analysis, we assessed safety and efficacy outcomes in all patients who had received treatment and been followed up for at least 18 months post-treatment on June 1, 2015. This trial is registered with ClinicalTrials.gov , number NCT01560182. Findings Between April, 2010, and February, 2013, we had enrolled nine children with a diagnosis of early-onset disease (six had late-infantile disease, two had early-juvenile disease, and one had early-onset disease that could not be definitively classified). At the time of analysis all children had survived, with a median follow-up of 36 months (range 18–54). The most commonly reported adverse events were cytopenia (reported in all patients) and mucositis of different grades of severity (in five of nine patients grade 3 in four of five patients). No serious adverse events related to the medicinal product were reported. Stable, sustained engraftment of gene-corrected HSCs was observed (a median of 60·4% range 14·0–95·6 lentiviral vector-positive colony-forming cells across follow-up) and the engraftment level was stable during follow-up; engraftment determinants included the duration of absolute neutropenia and the vector copy number of the medicinal product. A progressive reconstitution of ARSA activity in circulating haemopoietic cells and in the cerebrospinal fluid was documented in all patients in association with a reduction of the storage material in peripheral nerve samples in six of seven patients. Eight patients, seven of whom received treatment when presymptomatic, had prevention of disease onset or halted disease progression as per clinical and instrumental assessment, compared with historical untreated control patients with early-onset disease. GMFM scores for six patients up to the last follow-up showed that gross motor performance was similar to that of normally developing children. The extent of benefit appeared to be influenced by the interval between HSC-GT and the expected time of disease onset. Treatment resulted in protection from CNS demyelination in eight patients and, in at least three patients, amelioration of peripheral nervous system abnormalities, with signs of remyelination at both sites. Interpretation Our ad-hoc findings provide preliminary evidence of safety and therapeutic benefit of HSC-GT in patients with early-onset metachromatic leukodystrophy who received treatment in the presymptomatic or very early-symptomatic stage. The results of this trial will be reported when all 20 patients have achieved 3 years of follow-up. Funding Italian Telethon Foundation and GlaxoSmithKline.
HIV-1 insertions targeting BACH2 or MLK2 are enriched and persist for decades in hematopoietic cells from patients under combination antiretroviral therapy. However, it is unclear how these ...insertions provide such selective advantage to infected cell clones. Here, we show that in 30/87 (34%) patients under combination antiretroviral therapy, BACH2, and STAT5B are activated by insertions triggering the formation of mRNAs that contain viral sequences fused by splicing to their first protein-coding exon. These chimeric mRNAs, predicted to express full-length proteins, are enriched in T regulatory and T central memory cells, but not in other T lymphocyte subsets or monocytes. Overexpression of BACH2 or STAT5B in primary T regulatory cells increases their proliferation and survival without compromising their function. Hence, we provide evidence that HIV-1-mediated insertional activation of BACH2 and STAT5B favor the persistence of a viral reservoir in T regulatory cells in patients under combination antiretroviral therapy.HIV insertions in hematopoietic cells are enriched in BACH2 or MLK2 genes, but the selective advantages conferred are unknown. Here, the authors show that BACH2 and additionally STAT5B are activated by viral insertions, generating chimeric mRNAs specifically enriched in T regulatory cells favoring their persistence.
Bioinformatics tools designed to identify lentiviral or retroviral vector insertion sites in the genome of host cells are used to address the safety and long-term efficacy of hematopoietic stem cell ...gene therapy applications and to study the clonal dynamics of hematopoietic reconstitution. The increasing number of gene therapy clinical trials combined with the increasing amount of Next Generation Sequencing data, aimed at identifying integration sites, require both highly accurate and efficient computational software able to correctly process "big data" in a reasonable computational time.
Here we present VISPA2 (Vector Integration Site Parallel Analysis, version 2), the latest optimized computational pipeline for integration site identification and analysis with the following features: (1) the sequence analysis for the integration site processing is fully compliant with paired-end reads and includes a sequence quality filter before and after the alignment on the target genome; (2) an heuristic algorithm to reduce false positive integration sites at nucleotide level to reduce the impact of Polymerase Chain Reaction or trimming/alignment artifacts; (3) a classification and annotation module for integration sites; (4) a user friendly web interface as researcher front-end to perform integration site analyses without computational skills; (5) the time speedup of all steps through parallelization (Hadoop free).
We tested VISPA2 performances using simulated and real datasets of lentiviral vector integration sites, previously obtained from patients enrolled in a hematopoietic stem cell gene therapy clinical trial and compared the results with other preexisting tools for integration site analysis. On the computational side, VISPA2 showed a > 6-fold speedup and improved precision and recall metrics (1 and 0.97 respectively) compared to previously developed computational pipelines. These performances indicate that VISPA2 is a fast, reliable and user-friendly tool for integration site analysis, which allows gene therapy integration data to be handled in a cost and time effective fashion. Moreover, the web access of VISPA2 ( http://openserver.itb.cnr.it/vispa/ ) ensures accessibility and ease of usage to researches of a complex analytical tool. We released the source code of VISPA2 in a public repository ( https://bitbucket.org/andreacalabria/vispa2 ).
Abstract
High transduction rates of viral vectors in gene therapies (GT) and experimental hematopoiesis ensure a high frequency of gene delivery, although multiple integration events can occur in the ...same cell. Therefore, tracing of integration sites (IS) leads to mis-quantification of the true clonal spectrum and limits safety considerations in GT. Hence, we use correlations between repeated measurements of IS abundances to estimate their mutual similarity and identify clusters of co-occurring IS, for which we assume a clonal origin. We evaluate the performance, robustness and specificity of our methodology using clonal simulations. The reconstruction methods, implemented and provided as an R-package, are further applied to experimental clonal mixes and preclinical models of hematopoietic GT. Our results demonstrate that clonal reconstruction from IS data allows to overcome systematic biases in the clonal quantification as an essential prerequisite for the assessment of safety and long-term efficacy of GT involving integrative vectors.
Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a ...case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors.
Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned ...exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.
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