A case of T-cell acute lymphoblastic leukemia in retroviral gene therapy for ADA-SCID.

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.

Circulating hematopoietic stem/progenitor cell subsets contribute to human hematopoietic homeostasis.

ABSTRACT: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients' clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18).

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells.

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

InCliniGene enables high-throughput and comprehensive in vivo clonal tracking toward clinical genomics data integration.

High-throughput clonal tracking in patients under hematopoietic stem cell gene therapy with integrating vector is instrumental in assessing bio-safety and efficacy. Monitoring the fate of millions of transplanted clones and their progeny across differentiation and proliferation over time leverages the identification of the vector integration sites, used as surrogates of clonal identity. Although γ-tracking retroviral insertion sites (γ-TRIS) is the state-of-the-art algorithm for clonal identification, the computational drawbacks in the tracking algorithm, based on a combinatorial all-versus-all strategy, limit its use in clinical studies with several thousands of samples per patient. We developed the first clonal tracking graph database, InCliniGene (https://github.com/calabrialab/InCliniGene), that imports the output files of γ-TRIS and generates the graph of clones (nodes) connected by arches if two nodes share common genomic features as defined by the γ-TRIS rules. Embedding both clonal data and their connections in the graph, InCliniGene can track all clones longitudinally over samples through data queries that fully explore the graph. This approach resulted in being highly accurate and scalable. We validated InCliniGene using an in vitro dataset, specifically designed to mimic clinical cases, and tested the accuracy and precision. InCliniGene allows extensive use of γ-TRIS in large gene therapy clinical applications and naturally realizes the full data integration of molecular and genomics data, clinical and treatment measurements and genomic annotations. Further extensions of InCliniGene with data federation and with application programming interface will support data mining toward precision, personalized and predictive medicine in gene therapy. Database URL:  https://github.com/calabrialab/InCliniGene.

Intrathymic AAV delivery results in therapeutic site-specific integration at TCR loci in mice.

Adeno-associated virus (AAV) vectors have been successfully exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector, but it has been shown to integrate within the host cell genome after the generation of double-strand DNA breaks or nicks. Although AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T-cell progenitors and long-term T-cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T cells and compared these with liver and brain tissue from treated mice. Notably, we found that although AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T cells were clustered within the T-cell receptor α, ß, and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAGs) during variable, diversity, and joining recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.

IL-3-zetakine combined with a CD33 costimulatory receptor as a dual CAR approach for safer and selective targeting of AML.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T-cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the 2 most widely overexpressed leukemic stem cell biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced dual-CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-induced killer (CIK) cells, coexpressing a first-generation low affinity anti-CD123 interleukin-3-zetakine (IL-3z) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR), demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on hematopoietic stem and progenitor cells and endothelial cells. The proposed optimized dual-CAR cytokine-induced killer cell strategy could offer the opportunity to unleash the potential of specifically targeting CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

ISAnalytics enables longitudinal and high-throughput clonal tracking studies in hematopoietic stem cell gene therapy applications.

Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies.

Prolonged XPO1 inhibition is essential for optimal antileukemic activity in NPM1-mutated AML.

NPM1 is the most frequently mutated gene in adults with acute myeloid leukemia (AML). The interaction between mutant NPM1 (NPM1c) and exportin-1 (XPO1) causes aberrant cytoplasmic dislocation of NPM1c and promotes the high expression of homeobox (HOX) genes, which is critical for maintaining the leukemic state of NPM1-mutated cells. Although there is a rationale for using XPO1 inhibitors in NPM1-mutated AML, selinexor administered once or twice per week did not translate into clinical benefit in patients with NPM1 mutations. Here, we show that this dosing strategy results in only a temporary disruption of the XPO1-NPM1c interaction, limiting the efficacy of selinexor. Because the second-generation XPO1 inhibitor eltanexor can be administered more frequently, we tested the antileukemic activity of prolonged XPO1 inhibition in NPM1-mutated AML models. Eltanexor caused irreversible HOX downregulation, induced terminal AML differentiation, and prolonged the survival of leukemic mice. This study provides essential information for the appropriate design of clinical trials with XPO1 inhibitors in NPM1-mutated AML.

SiCoDEA: A Simple, Fast and Complete App for Analyzing the Effect of Individual Drugs and Their Combinations.

The administration of combinations of drugs is a method widely used in the treatment of different pathologies as it can lead to an increase in the therapeutic effect and a reduction in the dose compared to the administration of single drugs. For these reasons, it is of interest to study combinations of drugs and to determine whether a specific combination has a synergistic, antagonistic or additive effect. Various mathematical models have been developed, which use different methods to evaluate the synergy of a combination of drugs. We have developed an open access and easy to use app that allows different models to be explored and the most fitting to be chosen for the specific experimental data: SiCoDEA (Single and Combined Drug Effect Analysis). Despite the existence of other tools for drug combination analysis, SiCoDEA remains the most complete and flexible since it offers options such as outlier removal or the ability to choose between different models for analysis. SiCoDEA is an easy to use tool for analyzing drug combination data and to have a view of the various steps and offer different results based on the model chosen.

Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.).

Retrieval of vector integration sites from cell-free DNA.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML.

Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies.

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice.

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor-/- knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor-/-/Dnmt3a-/- knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.

ARPIR: automatic RNA-Seq pipelines with interactive report.

BACKGROUND: RNA-Seq is an increasing used methodology to study either coding and non-coding RNA expression. There are many software tools available for each phase of the RNA-Seq analysis and each of them uses different algorithms. Furthermore, the analysis consists of several steps regarding alignment (primary-analysis), quantification, differential analysis (secondary-analysis) and any tertiary-analysis and can therefore be time-consuming to deal with each step separately, in addition to requiring a computer knowledge. For this reason, the development of an automated pipeline that allows the entire analysis to be managed through a single initial command and that is easy to use even for those without computer skills can be useful. Faced with the vast availability of RNA-Seq analysis tools, it is first of all necessary to select a limited number of pipelines to include. For this purpose, we compared eight pipelines obtained by combining the most used tools and for each one we evaluated peak of RAM, time, sensitivity and specificity. RESULTS: The pipeline with shorter times, lower consumption of RAM and higher sensitivity is the one consisting in HISAT2 for alignment, featureCounts for quantification and edgeR for differential analysis. Here, we developed ARPIR, an automated pipeline that recurs by default to the cited pipeline, but it also allows to choose, between different tools, those of the pipelines having the best performances. CONCLUSIONS: ARPIR allows the analysis of RNA-Seq data from groups undergoing different treatment allowing multiple comparisons in a single launch and can be used either for paired-end or single-end analysis. All the required prerequisites can be installed via a configuration script and the analysis can be launched via a graphical interface or by a template script. In addition, ARPIR makes a final tertiary-analysis that includes a Gene Ontology and Pathway analysis. The results can be viewed in an interactive Shiny App and exported in a report (pdf, word or html formats). ARPIR is an efficient and easy-to-use tool for RNA-Seq analysis from quality control to Pathway analysis that allows you to choose between different pipelines.

γ-TRIS: a graph-algorithm for comprehensive identification of vector genomic insertion sites.

SUMMARY: Retroviruses and their vector derivatives integrate semi-randomly in the genome of host cells and are inherited by their progeny as stable genetic marks. The retrieval and mapping of the sequences flanking the virus-host DNA junctions allows the identification of insertion sites in gene therapy or virally infected patients, essential for monitoring the evolution of genetically modified cells in vivo. However, since ∼30% of insertions land in low complexity or repetitive regions of the host cell genome, they cannot be correctly assigned and are currently discarded, limiting the accuracy and predictive power of clonal tracking studies. Here, we present γ-TRIS, a new graph-based genome-free alignment tool for identifying insertion sites even if embedded in low complexity regions. By using γ-TRIS to reanalyze clinical studies, we observed improvements in clonal quantification and tracking. AVAILABILITY AND IMPLEMENTATION: Source code at https://bitbucket.org/bereste/g-tris. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically Relevant Ex Vivo Gene Therapy Settings.

Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit ex vivo LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow-derived CD34+ cells while better preserving their engraftment capacity in vivo. CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G0 fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels in vivo. Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC ex vivo culture time.

Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia.

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (ß+) or absent (ß0) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life1. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease2. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß0 or severe ß+ mutations in a phase 1/2 trial ( NCT02453477 ) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6-76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10-1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.

VISPA2: a scalable pipeline for high-throughput identification and annotation of vector integration sites.

BACKGROUND: 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. RESULTS: 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). CONCLUSIONS: 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 ).

Preclinical Testing of the Safety and Tolerability of Lentiviral Vector-Mediated Above-Normal Alpha-L-Iduronidase Expression in Murine and Human Hematopoietic Cells Using Toxicology and Biodistribution Good Laboratory Practice Studies.

In order to support the clinical application of hematopoietic stem cell (HSC) gene therapy for mucopolysaccharidosis I (MPS I), biosafety studies were conducted to assess the toxicity and tumorigenic potential, as well as the biodistribution of HSCs and progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of the alpha-iduronidase (IDUA) gene, which is mutated in MPS I patients. To this goal, toxicology and biodistribution studies were conducted, employing Good Laboratory Practice principles. Vector integration site (IS) studies were applied in order to predict adverse consequences of vector gene transfer and to obtain HSC-related information. Overall, the results obtained in these studies provided robust evidence to support the safety and tolerability of high-efficiency LV-mediated gene transfer and above-normal IDUA enzyme expression in both murine and human HSPCs and their in vivo progeny. Taken together, these investigations provide essential safety data to support clinical testing of HSC gene therapy in MPS I patients. These studies also underline criticisms associated with the use of currently available models, and highlight the value of surrogate markers of tumorigenicity that may be further explored in the future. Notably, biological evidence supporting the efficacy of gene therapy on MPS I disease and its feasibility on patients' HSCs were also generated, employing clinical-grade LVs. Finally, the clonal contribution of LV-transduced HSPCs to hematopoiesis along serial transplantation was quantified in a minimum of 200-300 clones, with the different level of repopulating cells in primary recipients being reflected in the secondary.