SCID-X1 Gene Therapy Research Articles

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Gene therapy for severe combined immunodeficiencies and beyond.

Ex vivo retrovirally mediated gene therapy has been shown within the last 20 yr to correct the T cell immunodeficiency caused by γc-deficiency (SCID X1) and adenosine deaminase (ADA) deficiency. The rationale was brought up by the observation of the revertant of SCIDX1 and ADA deficiency as a kind of natural gene therapy. Nevertheless, the first attempts of gene therapy for SCID X1 were associated with insertional mutagenesis causing leukemia, because the viral enhancer induced transactivation of oncogenes. Removal of this element and use of a promoter instead led to safer but still efficacious gene therapy. It was observed that a fully diversified T cell repertoire could be generated by a limited set (<1,000) of progenitor cells. Further advances in gene transfer technology, including the use of lentiviral vectors, has led to success in the treatment of Wiskott-Aldrich syndrome, while further applications are pending. Genome editing of the mutated gene may be envisaged as an alternative strategy to treat SCID diseases.

Enhanced Transduction Lentivector Gene Therapy for Treatment of Older Patients with X-Linked Severe Combined Immunodeficiency

Enhanced Transduction Lentivector Gene Therapy for Treatment of Older Patients with X-Linked Severe Combined Immunodeficiency

Engineering Naturally Occurring CD7 Negative Cells for the Immunotherapy of CD7 Positive Leukemia

Background: CD7 has emerged as a promising target for the adoptive immunotherapy with T-cells expressing chimeric antigen receptors (CAR T-cells) of CD7+ T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). However, expressing CD7 CARs in T-cells results in fratricide due to high expression of CD7 in most T-cells. While investigators have developed strategies to overcome this limitation by additional genetic modifications of CD7 CAR T-cells, the goal of this project was to explore the feasibility of selecting and genetically modifying naturally occurring CD7 negative (CD7-) T cells for the adoptive immunotherapy of CD7+ leukemia. Methods: CD7- T-cells were isolated from PBMCs using a 2-step magnetic bead depletion/selection procedure (CD7 depletion followed by selection of CD3+ T cells from the CD7- fraction). Non-selected T-cells (bulk T-cells), CD7+ and CD7- T cells were activated and transduced with a retroviral vector encoding a second-generation CD7 CAR with a CD28 costimulatory endodomain, and expanded with IL7 and IL15. The effector function of CD7- T-cells expressing CD7 CARs (CD7 CARCD7- T cells) was assessed in vitro as well as in xenograft models. Results: To assess the feasibility of our approach, we first determined the frequency of CD7- T-cells in PBMCs. On average, 4.7 % of T cells were CD7- (range: 2% - 12.3%; N=22), and we successfully selected these cells from bulk PBMCs with a combined CD7 depletion/CD3 selection procedure. We genetically modified CD7-, CD7+ and bulk T cellsto express CD7 CARs (CD7 CARCD7-, CD7 CARCD7+, CD7 CARBulk). Transduction efficiencies ranged from 31% to 75% (± 5%) for each T-cell population. Post transduction, CD7 CARCD7- T-cells did not undergo fratricide and had similar expansion kinetics (N=6, p=ns) in comparison to non-transduced (NT) T-cell cultures (NT CD7-, NT CD7+, NT bulk). In contrast, CD7 CARCD7+or CD7 CARBulk T-cells underwent fratricide and did not expand (N=6, p&amp;lt;0.0001). CD7- T-cells (NT and CD7 CARCD7-) had a predominantly CD4+ effector memory phenotype at day 7 and 14 of culture. To assess the effector function of CD7 CARCD7- T-cells, we co-cultured them with CD7+ T-ALL cell lines (CCRF, MOLT3). CD7 CARCD7- T-cells recognized CD7+ targets in contrast to CD7- targets (BV173, Daudi) as evidenced by significant (N=6, p&amp;lt;0.0001) IFN-γ and IL-2 production. Control CAR T-cells (CD19 CARCD7-) did not recognize CD7+ target cells, confirming specificity. CD7 CARCD7- T-cells also had potent cytolytic activity against CD7+ targets in cytotoxicity assays. To assess in vivo the anti-tumor activity of CD7 CARCD7- T-cells, we used a NSG mouse xenograft model with CCRF cells, genetically modified to express firefly luciferase (CCRF.ffluc) to allow for serial bioluminescence imaging. A single infusion of CD7 CARCD7- T-cells had potent anti-leukemia activity as judged by serial imaging resulting in a significant survival (p&amp;lt;0.003) advantage in comparison to control mice. Conclusion: We have successfully generated CD7 CARCD7- T-cells from peripheral blood CD7- T-cells. CD7 CARCD7- T-cells had a predominantly CD4+ effector memory phenotype, and potent anti-leukemia activity in vitro and in vivo. Thus, naturally occurring CD7- T cells may present a promising T-cell source for the cellular immunotherapy of CD7+ leukemia. Disclosures Langfitt: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Youngblood:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Gottschalk:NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; ASSISI fundation of Memphis: Research Funding; California Institute for Regenerative Medicine: Research Funding; ViraCyte: Consultancy; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Patents and patent applications in the fields of T-cell &amp; Gene therapy for cancer: Patents &amp; Royalties; TESSA Therapeutics: Other: Research Collaboration; Tidal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; EMD Serono: Honoraria; Merck: Consultancy; Inmatics: Membership on an entity's Board of Directors or advisory committees. Velasquez:St. Jude: Patents &amp; Royalties: Patent Applications in the Fields of Cell and Gene Therapy ; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

Neurocognitive Impairment Predicts Poor Transition Outcomes Among Patients with Sickle Cell Disease

Introduction: In the United States, most children with sickle cell disease (SCD) survive into adulthood and transfer from pediatric to adult-centered care. Cognitive deficits begin during childhood and are highly prevalent among individuals with SCD, potentially affecting their functional ability to establish adult care and navigate the new adult care environment. Lack of engagement in adult care can place youth with SCD at higher risk for care discontinuity and higher disease morbidity and mortality. The relationship between cognition and transition to adult care has not been examined. We hypothesized that better performance on measures of neurocognition were associated with decreased latency in initiating adult care, greater retention in adult care, and increased utilization of adult ambulatory services. As a secondary objective, we examined the relationship of environmental outcomes to transition outcomes. Methods: We included participants enrolled in the Sickle Cell Research and Intervention Program (SCCRIP; Hankins J. et al, Pediatric Blood and Cancer 2018), a longitudinal lifetime cohort study of individuals with SCD that monitors neurocognition. Participants were included if they underwent neurocognitive screening assessment in adolescence, prior to their transfer to adult care and if they satisfied their first appointment in adult care. The neurocognitive screening battery included measures of estimated global intelligence (Wechsler Abbreviated Scales of Intelligence, 2nd Ed; WASI-2) and sustained attention (Continuous Performance Test, 2nd Ed; CPT-2). Environmental factors included the Economic Hardship Index (EHI), guardian employment status while in pediatric care, and the number of persons living in the household. Use of adult ambulatory services was measured by the number of outpatient visits per patient-year. The association between cognitive performance and the latency from pediatric to adult care, adult care retention and environmental variables was examined using the 2-sample t test if the data were normally distributed or the Wilcoxon rank-sum test otherwise. Categorical variables were analyzed with the Chi-square test or Fisher's exact test. Transition outcomes were also analyzed as continuous variables using univariate linear regression. All reported p-values are two-sided. Results: Eighty adolescents with SCD ages 15-18 years at the time of their cognitive assessment (58% male, 63% HbSS/HbSβ0-thalassemia) were included; most transferred &amp;lt;6 months from the last pediatric visit Table 1). Of these 80 patients, 61 and 43 had sufficient follow-up time to examine their retention in adult care 12 and 24 months after transfer, respectively. Fifty out of the 61 patients (82%) remained in adult care &amp;gt; 12 months, and 31 of the 43 (72%) remained in adult care &amp;gt;24 months after their first adult visit. Higher Full-Scale IQ was associated with establishing adult care ≤2 months from last pediatric visit (Table 1; Figure 1A, 1B). Belonging to families with fewer children, smaller households and a higher WASI-2 Verbal Comprehension Index were associated with establishing adult care ≤6 months from last pediatric visit. Better CPT-2 Commissions performance (less attention deficit) was associated with increased adult care retention at 12 and 24 months (Table 2; Figure 1C,1D). Having a working guardian was associated with less retention at 12 months (p=0.01), whereas having an unemployed primary guardian was associated with greater retention at 24 months (p=0.02). Further, an employed guardian was associated with greater utilization of adult ambulatory services (p=0.01). EHI was not significantly related to transition outcomes. No relationship was found between adult ambulatory services and neurocognitive assessment. Conclusion: Neurocognitive deficit (lower IQ and attention deficits) may decrease short and long-term engagement in adult care among youth with SCD as demonstrated by longer latency periods between pediatric and adult care and shorter adult care retention. Socio-economic factors may also play a role in transition outcomes but require further investigation. Investigation of disease modifying therapies that preserve cognitive function should be prioritized. Interventions that account for patients' cognitive level and their environment should be considered in the individualization of transition plans. Disclosures King: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novimmune: Research Funding; Amphivena Therapeutics: Research Funding; Incyte: Consultancy; Tioma Therapeutics (formerly Vasculox, Inc.):: Consultancy; Cell Works: Consultancy; Bioline: Consultancy; Celgene: Consultancy; RiverVest: Consultancy; WUGEN: Equity Ownership. Wang:Agios Pharmaceuticals: Consultancy; Novartis: Consultancy. Zhao:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Kang:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Hankins:National Committee for Quality Assurance: Consultancy; NHLBI: Research Funding; Global Blood Therapeutics: Research Funding; Novartis: Research Funding; LYNKS Foundation: Research Funding; NHLBI: Honoraria; ASPHO: Honoraria; Bluebird Bio: Consultancy.

Transcranial Doppler Velocities Conversion Rate Based on Increasing Hemoglobin Concentration: Analysies from the SCCRIP Cohort Study

Transcranial Doppler Velocities Conversion Rate Based on Increasing Hemoglobin Concentration: Analysies from the SCCRIP Cohort Study

Longitudinal Effect of Hydroxyurea Therapy on Left Ventricular Diastolic Function in Sickle Cell Anemia

Longitudinal Effect of Hydroxyurea Therapy on Left Ventricular Diastolic Function in Sickle Cell Anemia

Evaluation of Factors Influencing Health Literacy in Adolescents and Adults with Sickle Cell Disease

Introduction In the United States 36% of adults have limited health literacy, which is associated with poor health outcomes (Kutner et al. National Assessment of Adult Literacy.2006). Health literacy is defined as "the capacity to obtain, process and understand basic health information and services needed to make appropriate health decisions" (Nielsen-Bohlman et al. National Academies Press.2004). Health literacy is understudied in sickle cell disease (SCD), a genetic disease affecting a minority population in the United States. SCD Preventive treatment requires patient engagement, therefore health literacy is an important determinant of health outcomes in SCD. Previous studies reported Health literacy in adolescents with SCD is suboptimal (Perry et al. J Pediatr Nur.2017), but few studies have investigated factors influencing health literacy in this population. Health literacy can be impacted by many factors such as cognition, socioeconomic status, education level and gender. This study evaluated health literacy level and factors that influenced Health literacy in adolescents and adults with SCD. In addition, we assessed the relationship of health literacy level and Hydroxyurea therapy use, one of only two FDA-approved disease-modifying therapies for SCD. Methods This was a cross- sectional study of adolescents and adults with SCD performed at St. Jude Children's Research hospital and St. Louis Children's hospital. Adolescents and adults ages 15 to 45 years completed a health literacy assessment using the newest vital sign (NVS). NVS is a validated tool with 6 questions that tests reading, mathematics and comprehension based on a nutrition label (Weiss et al. Ann Fam Med.2005). Score of 0-1 suggests high likelihood of limited health literacy, 2-3 indicates possibility of limited health literacy and 4-6 almost always suggests adequate health literacy. Self-reported demographic information was collected such as educational level, household income, age, sex, race. Additionally, SCD genotypes and hydroxyurea utilization were confirmed through chart review. Results A random sample of 125 adolescents and adults with SCD was evaluated (Table 1): 34 (28.1%) had Hb SC or Hb Sβ+-thalassemia, 87(71.9%) had Hgb SS or Hgb Sβ0-thalassemia, and 4 participants had unknown or other genotypes. Limited health literacy was prevalent, only 40 (32%) of subjects had adequate literacy, median NVS score was 2 for all participants. Participants with Hgb SS/Hgb Sβ0-thalassemia were more likely to be on hydroxyurea (p&lt;0.001, Chi-square test). NVS scores in subjects with less severe genotype were not significantly different compared to more severe genotypes (p=0.2, Man-Whitney-Wilcoxon test/MWW). There was no difference in health literacy scores between those on hydroxyurea and not on hydroxyurea (p=0.24, MWW test), however despite low health literacy being prevalent, 85 (68%) subjects with Hb SS or HbSβ0-thalassemia were on hydroxyurea. Income level was significantly different between patient on hydroxyurea not on hydroxyurea (p= 0.034, Fisher's exact test). In addition, patient whose income was $35000 or more were found to have a higher NVS score (p=0.012, Kruskal-Wallis test) (figure 1). Subjects with high NVS scores were significantly older (p=0.026, Analysis of variance test), over all education was significantly associated with health literacy (p=0.003, Fisher's exact). Conclusion This study shows a low prevalence of adequate health literacy amongst adolescents and adults with SCD. Our data corroborate previous literature which has reported limited health literacy in this population as well adds granularity to the factors that may influence this suboptimal outcome. There was a statistically significant association between health literacy, income level and education level in adolescents and young adults with SCD. Further, health literacy was not significantly associated with sickle cell genotype or hydroxyurea use. This study highlights the social determinants of health and specific need to address health literacy in SCD patients. To optimize the care of adolescents and young adults with SCD, targeted multimodal interventions that are tailored to low health literacy levels should be employed to reduce morbidity and mortality in this vulnerable population. Disclosures Kang: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. King:Amphivena Therapeutics: Research Funding; Bioline: Consultancy; Celgene: Consultancy; Cell Works: Consultancy; Incyte: Consultancy; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novimmune: Research Funding; RiverVest: Consultancy; Tioma Therapeutics (formerly Vasculox, Inc.):: Consultancy; WUGEN: Equity Ownership. Zhao:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Hankins:NHLBI: Research Funding; National Committee for Quality Assurance: Consultancy; Global Blood Therapeutics: Research Funding; NHLBI: Honoraria; ASPHO: Honoraria; LYNKS Foundation: Research Funding; Novartis: Research Funding; Bluebird Bio: Consultancy.

Progression of Central Nervous System Vasculopathy in Young Adults with Sickle Cell Anemia

Progression of Central Nervous System Vasculopathy in Young Adults with Sickle Cell Anemia

CRISPR-Cas9 Genome Editing of γ-Globin Promoters in Human Hematopoietic Stem Cells to Induce Erythrocyte Fetal Hemoglobin for Treatment of β-Hemoglobinopathies

Induction of fetal hemoglobin (HbF, α2γ2) via genome editing-mediated disruption of DNA regulatory elements that repress expression of γ-globin genes (HBG1 and HBG2) is a promising therapeutic strategy for b-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia. Optimal technical approaches and safety profiles are yet to be fully defined. We used CRISPR/Cas9 to target a DNA repressor element near the distal CCAAT box of the HBG1/HBG2 promoters. This region contains a "TGACC" motif recognized by BCL11A, a transcriptional repressor protein that regulates γ-to-β globin switch after birth. Rare germline variants at or near this motif are associated with hereditary persistence of fetal hemoglobin, a benign genetic condition that alleviates the clinical manifestations of co-inherited b-hemoglobinopathies. Previously, we showed that transduction of human CD34+ cells with lentiviral vector encoding Cas9 and guideRNA (gRNA) targeting the HBG1/HBG2 promoter caused induction of HbF in red blood cell (RBC) progeny generated in vitro (Traxler et. al, Nature Medicine v22,2016). Here we present a clinically tractable approach for disrupting the HBG1/HBG2 BCL11A binding site in human hematopoietic stem cells (HSCs). Electroporation of Cas9:gRNA ribonucleoprotein (RNP) complex into healthy or SCD donor CD34+ cells resulted in up to 80% on-target insertion-deletion (indel) mutations and 35% HbF in erythroid progeny generated in vitro. Sixteen to 17 weeks after transplantation of gene edited CD34+ cells into immunodeficient NBSGW mice, up to 75% of donor CD34+ cells in recipient bone marrow contained on-target indels, demonstrating efficient modification of repopulating human HSCs. No differences in CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages were observed between edited and control cells. Moreover, up to 78% of gene edited erythroid cells stained with anti-HbF antibody ("F-cells") compared to 15% in control erythroid cells, suggesting a "pan-cellular" pattern of HbF expression after editing. Strikingly, human donor-derived erythroid cells in recipient bone marrow expressed up to 40% HbF compared to 3% HbF in controls. Although the editing frequencies of HBG1 and HBG2 promoters varied between different donor CD34+ cells, an engineered variant of Cas9 containing 3 nuclear localization sequences (Wu et. al,Nature Medicine v25, 2019) edited repopulating HSCs more efficiently and consistently than conventional Cas9 with two nuclear localization signals. Simultaneous on-target RNP-induced DSBs at both HBG1 and HBG2 can result in the deletion of the intervening 4.9-kb region, leaving a single hybrid gene with HBG2 promoter sequences fused to the downstream HBG1 gene. We detected this deletion in approximately 30% of edited cells, with no associated decline in HbF expression determined by clonal analysis of erythroid colonies. No off-target mutations were detected by targeted sequencing of the 26 top candidate sites identified by CIRCLE-seq, an in vitro genome-scale method for detecting Cas9 activity. Analysis of gene edited human donor cells purified from mouse bone marrow showed no chromosomal rearrangements by G-banding (n=20) or fluorescence in situ hybridization with a probe located distal to the HBG1/HBG2 loci (n=225). Taken together, our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a CRISPR-Cas9 genome editing approach to induce HbF for treating hemoglobinopathies. Figure. Gene editing of the HBG1/HBG2 promoters in HSCs and HbF induction of erythroid progeny in vivo. Plerixafor-mobilized CD34+ cells from an individual with SCD were edited with RNP and transplanted into NBSGW mice, which were analyzed 16-17 weeks later. A. On-target indel frequency before (Pre) and after bone marrow transplantation (BM). The black bars represent a 13-nucleotide deletion associated with human hereditary persistence of fetal hemoglobin. B. Human erythroblasts and reticulocytes derived from RNP-edited and non-edited Control CD34+ donor cells. Scale bar = 10 mm. C. HbF immunostaining control and RNP edited erythroid cells in recipient bone marrow assessed by flow cytometry. D. %HbF protein in hemolysates of control (C) and RNP edited erythroid cells assessed by ion-exchange HPLC. n= 3 biological replicates. **** P &lt; 0.0001. Figure Disclosures Metais: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Sharma:Doris Duke Foundation: Research Funding; Vertex Pharmaceuticals: Other: Study PI. Weiss:Beam Therapeutics: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy.

T cell dynamics and response of the microbiota after gene therapy to treat X-linked severe combined immunodeficiency

BackgroundMutation of the IL2RG gene results in a form of severe combined immune deficiency (SCID-X1), which has been treated successfully with hematopoietic stem cell gene therapy. SCID-X1 gene therapy results in reconstitution of the previously lacking T cell compartment, allowing analysis of the roles of T cell immunity in humans by comparing before and after gene correction.MethodsHere we interrogate T cell reconstitution using four forms of high throughput analysis. (1) Estimation of the numbers of transduced progenitor cells by monitoring unique positions of integration of the therapeutic gene transfer vector. (2) Estimation of T cell population structure by sequencing of the recombined T cell receptor (TCR) beta locus. (3) Metagenomic analysis of microbial populations in oropharyngeal, nasopharyngeal, and gut samples. (4) Metagenomic analysis of viral populations in gut samples.ResultsComparison of progenitor and mature T cell populations allowed estimation of a minimum number of cell divisions needed to generate the observed populations. Analysis of microbial populations showed the effects of immune reconstitution, including normalization of gut microbiota and clearance of viral infections. Metagenomic analysis revealed enrichment of genes for antibiotic resistance in gene-corrected subjects relative to healthy controls, likely a result of higher healthcare exposure.ConclusionsThis multi-omic approach enables the characterization of multiple effects of SCID-X1 gene therapy, including T cell repertoire reconstitution, estimation of numbers of cell divisions between progenitors and daughter T cells, normalization of the microbiome, clearance of microbial pathogens, and modulations in antibiotic resistance gene levels. Together, these results quantify several aspects of the long-term efficacy of gene therapy for SCID-X1. This study includes data from ClinicalTrials.gov numbers NCT01410019, NCT01175239, and NCT01129544.

Preclinical Development of Gene Therapy for X-Linked Severe Combined Immunodeficiency (SCID-X1)

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the interleukin-2 receptor γ chain (IL2RG), and is characterized by lack of response to common γ-chain-dependent cytokines and profound defects in T-, B- and NK-cell functions. Previous gene therapy clinical trials based on autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with MLV-derived retroviral vectors expressing IL2RG in non-conditioned patients showed long-lasting restoration of T-cell immunity in most cases but resulted in vector-induced leukemia through enhancer-mediated insertional mutagenesis in 25% of patients. The use of an enhancer-less MLV vector to deliver the IL2RG gene caused no adverse events while retaining a significant clinical benefit. To increase the efficacy of gene therapy and reduce at the same time the probability of genotoxic insertional events, we developed a third-generation lentiviral vector carrying a codon-optimized human IL2RG cDNA under the control of the human EF1α-S promoter. The vector was VSV-G-pseudotyped and produced by transient transfection of 293T cells, followed by ion-exchange chromatography, tangential-flow filtration and formulation in HSC growth medium. Replacement of the native IL2RG open reading frame by a codon-optimized sequence resulted in a 3-fold increase in mRNA expression and a 1.5-fold increase in IL2RG protein expression per integrated vector copy. The performance of the vector was demonstrated in vitro by the restoration of a normal level of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3, and by high efficiency (>80%) transduction of human mobilized CD34+ HSPCs and transgene expression with no impact on viability or clonogenic capacity.An in vitro immortalization assay (IVIM) showed a safe genotoxic profile, while the in vivo safety and efficacy of the vector was tested it in a preclinical model of SCID-X1 gene therapy based on transplantation of genetically corrected Lin- cells from IL2rg-/- donor mice into lethally-irradiated IL2rg-/--Rag2-/- recipients. The study showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts in bone marrow and peripheral blood, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals, six months after transplantation.An extensive insertion site analysis was carried out in bone marrow, thymus and peripheral blood of individual or groups of animals to detect the presence of any clonal abnormality or skewing, and any deviation from a normal lentiviral integration pattern. The study showed the expected genomic integration profile and no signs of clonal dominance of transduced cells. Interestingly, analysis of >100,000 integration sites in pre- and post-transplant murine cells showed that lentiviral vectors target at high frequency a substantially different set of genes compared to human CD34+ cells, uncovering the need for appropriate controls and at the same time the limits in the predictive power of mouse-based genotoxicity studies, particularly those addressing proto-oncogene activation and clonal dominance.These studies will enable a multicenter phase-I/II clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1. The clinical protocol is based on transplantation of autologous CD34+ HSPCs transduced with the EF1a-IL2RGco lentiviral vector in infants after non-myeloablative conditioning, and aims at stable and sustained restoration of both T- and B-cell immunity. DisclosuresWilliams:Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

425. Production of Gene Corrected T Cell Precursors for Therapy of SCID X1

Gene therapy for SCID and CID usually leads to a more efficient T-cell reconstitution as compared to HSCT from a mismatched related donor. However, as observed in ADA, SCID X1 and WAS gene therapy trials, T cell recovery is sometimes slow and associated with morbidity and mortality due to viral infections. In order to shorten post-transplant immunodeficiency and to reduce the frequency of these complications, one possible strategy is to transplant gene-corrected T-cell committed precursors. We have recently set up a protocol for transplanting in vitro-committed T-cell precursors able to seed the thymus and generate a wave of mature and polyclonal T-cells significantly faster than is usually observed. This protocol is based on a 3 to 7 day-culture of CD34+ cells in the presence of immobilized Notch ligand Delta-like-4 (DL-4), an adhesion molecule and a combination of cytokines; this culture system enables the in vitro generation of large amounts of T-cell precursor cells that (i) display the phenotypic and molecular signatures (i.e. gene expression pattern and TCR rearrangement pattern) of early thymic progenitor cells and (ii) have a high T-lymphopoietic potential in vitro and in vivo when transferred into NOD/SCPD/γc knock-out (NSG) mice (Reimann C, Stem Cells, 2012). The goal of the present project was to combine exposure to DL-4 and gene therapy approaches (DL-4/GT). For this purpose, gene correction of CD34+ cells from SCID-X1 patients was performed on DL-4 coated plates and the ex vivo transduced cells were either seeded on OP9/DL-1 culture or injected into NSG mice to determine their in vitro and in vivo capacity to generate mature and diverse T cells. In the DL-4/GT condition, the frequency and number of CD7+CD5+commited T-cell precursors was increased as compared to GT condition alone. Experiments to assess if the DL-4/GT condition accelerate T cell differentiation of gene corrected cells are currently ongoing. Altogether, our results suggest that a short exposure to the Notch ligand DL-4 might be an useful tool to generate large amounts of corrected T-cell precursors.

690. Development of a Clinical Lentiviral Vector for Gene Therapy of SCID-X1

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the interleukin-2 receptor γ chain (IL2RG), and is characterized by profound immunological defects caused by a partial or complete absence of T and NK cells and the presence of non-functional B cells. To overcome the safety issues raised by the use of MLV-based retroviral vectors in previous gene therapy clinical trials, we designed a SIN lentiviral vector (LV) carrying the codon-optimized human IL2RG cDNA under the control of the human EF1αS promoter and a mutated WPRE. Replacement of the native IL2RG open reading frame by a codon-optimized sequence resulted in a 3-fold increase in mRNA expression and a 1.5-fold increase in IL2RG protein expression per integrated vector copy. The vector was VSV-G-pseudotyped and produced by a new manufacturing process based on quadri-transfection of suspension-adapted 293T cells grown in serum-free conditions in 50- to 200-L bioreactors, purified by ion-exchange chromatography and concentrated by tangential-flow filtration. The efficacy of this vector was demonstrated in vitro by the restoration of a normal level of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3 and by high efficiency (81±7%) transduction of human mobilized CD34+ hematopoietic stem/progenitor cells with no impact on viability or clonogenic capacity. A biosafety evaluation study of the IL2RG LV in the murine model of the disease showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals. An in vitro assay (IVIM) showed a safe genotoxic profile, while insertion site analysis in transplanted mice revealed a standard lentiviral integration profile and no signs of clonal dominance. These studies will enable a multicenter phase-I/II clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1.

284. Long-Term Therapeutic Immune Reconstitution in XSCID Canine Model via In Vivo Foamy Virus Delivery of Common Gamma Chain

X-linked combined immunodeficiency disease (XSCID) is caused by mutation in the common gamma chain, γC (interleukin-2 receptor subunit gamma, IL2RG) in both humans and canines. It is characterized by the inability of T-cell development leading to absence of T-cells in peripheral blood, lack of T-cell mediated immune response, low IgA and IgG levels, and early infant mortality. In the 1990s, human XSCID clinical trials utilizing gamma-retroviral vectors to deliver the IL2RG gene caused leukemia in 5 out of 20 patients due to vector integration in or near proto-oncogenes. Recent studies showed Foamy virus based vectors as an excellent alternative for in vivo gene-therapy because it is non-pathogenic in humans while exhibiting increased serum stability and favorable integration pattern. Previously, we have demonstrated CD3+ T-cell reconstitution in the canine model via intravenous injection of foamy virus expressing human elongation factor-1 alpha promoter (Ef1α)-yC. Unfortunately, the treated animals contained a low number of gene corrected progenitors at a sub-therapeutic level. Here, we achieved long-term therapeutic immune-reconstitution by intravenous delivery of a human phosphoglycerate kinase promoter (Pgk)-mediated γC foamy viral vector into XSCID neonatal canines. Long-term (2 years) post-injection follow-up demonstrated therapeutic levels of CD3+ T-cell expansion. Within the T-cell population, gene correction with Pgk-γC stabilized at ~80%. We validated T-cell functionality by using spectratyping analysis, which exhibited a diverse repertoire of receptor gene rearrangement. Retroviral integration site analysis (RIS) indicated polyclonal contribution to the reconstituted T-cells. Immunoglobulin ELISA assays showed that IgA and IgG levels in peripheral blood are comparable to normal healthy controls. We immunized the gene-corrected canine recipients with bacteriophage ϕx174 and confirmed production of specific IgG antibodies, showing the ability for isotype switching in B-lymphocytes. Currently, the gene-corrected canines exhibit comparable health and physical attributes to normal controls. Furthermore, semen from the gene-corrected male canine was used via artificial insemination to produce a litter of viable offsprings. In summary, our data demonstrate that Pgk-γC foamy viral vector delivered long-term therapeutic gene correction in a large-animal model for XSCID gene therapy. Most importantly, these results indicate that in vivo Pgk-γC foamy vector administration is a viable option for long-term immune reconstitution in future XSCID human clinical trials.

Gene Therapy for X-Linked Severe Combined Immunodeficiency: Where Do We Stand?

More than 20 years ago, X-linked severe combined immunodeficiency (SCID-X1) appeared to be the best condition to test the feasibility of hematopoietic stem cell gene therapy. The seminal SCID-X1 clinical studies, based on first-generation gammaretroviral vectors, demonstrated good long-term immune reconstitution in most treated patients despite the occurrence of vector-related leukemia in a few of them. This gene therapy has successfully enabled correction of the T cell defect. Natural killer and B cell defects were only partially restored, most likely due to the absence of a conditioning regimen. The success of these pioneering trials paved the way for the extension of gene-based treatment to many other diseases of the hematopoietic system, but the unfortunate serious adverse events led to extensive investigations to define the retrovirus integration profiles. This review puts into perspective the clinical experience of gene therapy for SCID-X1, with the development and implementation of new generations of safer vectors such as self-inactivating gammaretroviral or lentiviral vectors as well as major advances in integrome knowledge.

Faster T-cell development following gene therapy compared with haploidentical HSCT in the treatment of SCID-X1

AbstractDuring the last decade, gene therapy via ex vivo gene transfer into autologous hematopoietic stem cells has emerged as a convincing therapy for severe combined immunodeficiency caused by ILR2G mutation (SCID-X1) despite the occurrence of genotoxicity caused by the integration of first-generation retroviral vectors. However, the place of gene therapy among the therapeutic armamentarium remains to be defined. We retrospectively analyze and compare clinical outcomes and immune reconstitution in 13 consecutive SCID-X1 patients having undergone haploidentical hematopoietic stem cell transplantation (HSCT) and 14 SCID-X1 patients treated with gene therapy over the same period at a single center level: the Necker Children's Hospital (Paris, France). Our results show a clear advantage in terms of T-cell development of gene therapy over HSCT with a mismatched donor. Patients treated with gene therapy display a faster T-cell reconstitution and a better long-term thymic output. Interestingly, this advantage of gene therapy vs haploidentical HSCT seems to be independent of the existence of clinical graft-versus-host disease in the latter condition. If data of safety are confirmed over the long term, gene therapy for SCID-X1 appears to be an equal, if not superior, alternative to haploidentical HSCT.

243. Pre-Clinical Development of Lentiviral Gene Therapy for X-Linked Severe Combined Immunodeficiency

X-linked severe combined immunodeficiency (SCID-X1, OMIM #300400) is caused by mutations in the gene encoding the interleukin-2 receptor γ-chain (IL2RG) resulting in a lack of response to common γ-chain-dependent cytokines, absence of T- and NK-cell development, and impairment of B-cell function. Previous SCID-X1 clinical trials based on MLV-based retroviral vectors expressing the IL2RG gene successfully restored immunity in most patients, demonstrating the efficacy of the gene therapy despite the occurrence on insertional T leukemias in some patients. To overcome these side effects, we decided to developed a SIN lentiviral-vector (LV) carrying a codon-optimized human IL2RG cDNA under the control of the human EF1α-S promoter, including a mutated WPRE. The vector was VSV-G-pseudotyped and produced by a new manufacturing process based on quadri-transfection of suspension-adapted 293T cells grown in bioreactors in serum-free conditions, purified by ion-exchange chromatography and concentrated by tangential-flow filtration. The activity of the LV was tested in vitro by transduction of the IL2RG-deficient EDR7 human T-cell line and cord blood-derived or peripheral blood mobilized CD34+ hematopoietic progenitors. The LV reconstituted normal levels of surface IL2RG in ED7R cells at an average VCN of 2 and 90% transduction efficiency. Transduction of CD34+ cells resulted in a VCN of 2.8±0.7 at a transduction efficiency of 82±9% by CFC assay with no impact on viability and cloning efficiency. Induction of aberrant splicing of genes harboring LV proviruses was assayed in EDR7 cell clones in which LV integrations were characterized by LM-PCR and sequencing. The LV caused abnormal splicing at low frequency between upstream exons and constitutive or cryptic proviral splice sites located in the LTR or at internal positions. To test the therapeutic efficacy and potential genotoxicity of the LV, we transplanted lethally-irradiated IL2RG-deficient mice with BM-derived, lin- cells transduced at 72±15% efficiency with a VCN of 2.5±1.1. Transplanted mice are being tested at 3 and 6 months post-transplantation for restoration of T- and B-cell function. Integration site analysis will be performed to monitor LV integration and analyze the clonal repertoire of transduced BM and peripheral blood cells. Potential genotoxicity is also assayed by an in vitro immortalization (IVIM) study on primary cells. These studies will enable a multicenter phase-I/II/registration clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1.

291. A Novel Safety Assay for Retroviral Vectors That Reproduces Lmo2 Proto-Oncogene Insertional Activation Events

Gene therapies for X-linked severe combined immunodeficiency (SCID-X1) and Wiskott-Aldrich syndrome (WAS) with gammaretroviral vectors have caused a number of cases of acute lymphoid leukemias due to insertional activation of oncogenes, mostly LMO2. Transduction of murine bone marrow hematopoietic cells and subsequent culture for myeloid immortalization or transplant into recipient mice detected the oncogenic activity of the spleen focus-forming virus (SFFV) vector, which was used in the WAS trial, but failed to detect the oncogenic activity of the SCID-X1 gene therapy vector MFG-gc. The lack of the most relevant Lmo2 insertion by gammaretroviral vectors in these assays demands more relevant and sensitive vector safety assays. It has been shown that murine early thymic progenitor cells (ETP), when cultured on OP9-DL1 stroma cells, progress through the DN1, DN2, DN3, DN4, DP and SP differentiation stages, and that overexpression of LMO2 in ETP cells caused a differentiation block at the DN2 stage (CD4-CD8-CD25+CD44+). We tested whether transduction of ETP cells with gammaretroviral or lentiviral vectors could reproduce Lmo2 integrations and induce DN2 differentiation block. We tested three different gamma-retroviral vectors MSCV-GFP, SFFV-GFP and MFG-gc and two lentiviral vectors Cl20-SFFV-mCherry and Cl20i4r-SFFV-mCherry. The two self-inactivating lentiviral vectors contain a single internal SFFV long terminal repeat but differ in that one contains the cHS400 chromatin insulators. We transduced ETP cells with these vectors and cultured them on OP9-DL1 cells for up to 40 days. The range of vector copy number was 1.07-10.78 for the MSCV-GFP vector, 0.24-1.22 for the SFFV-GFP vector, 0.59-7.98 for the MFG-gc vector, 3.97-13.04 for the Cl20-SFFV-mCherry vector and 4.57-18.21 for the Cl20i4r-SFFV-mCherry vector at days 10-11. Between days 20-35, a distinct DN2-blocked cell subpopulation was clearly detected by flow cytometry in all the 20 gamma-retroviral groups and also in 3/9 lentiviral groups. None of the 6 mock groups had any evidence of DN2 blockade. Insertion site mapping of sorted DN2 cells showed that in the 10 MSCV-GFP groups, 3 had Lmo2 insertions, 3 had Mef2c insertions, and 3 had both Lmo2 and Mef2c insertions; in the 6 SFFV-GFP groups, 3 had Lmo2 insertions; in the 4 MFG-gc groups, 3 had Lmo2 insertions; both Cl20-SFFV-mCherry groups had Mef2c insertions and the one Cl20i4r-SFFV-mCherry group had Lmo2 insertion. These insertions occurred either in the introns or within +/- 50kb window of the gene. Transplant of the DN2-blocked cells from selected MSCV-GFP groups into recipient mice led to acute lymphoid leukemias. These results show that the transduction of ETP cells and the resultant DN2 blockade represent a more relevant and sensitive assay for vector safety assessment. Our result also suggests that an ETP-like subpopulation may be present in the bone marrow of SCID-X1 and WAS patients and explain the enhanced propensity of these disorders to oncogenic transformations.

Clinical applications of gene therapy for primary immunodeficiencies.

Primary immunodeficiencies (PIDs) have represented a paradigmatic model for successes and pitfalls of hematopoietic stem cells gene therapy. First clinical trials performed with gamma retroviral vectors (γ-RV) for adenosine deaminase severe combined immunodeficiency (ADA-SCID), X-linked SCID (SCID-X1), and Wiskott-Aldrich syndrome (WAS) showed that gene therapy is a valid therapeutic option in patients lacking an HLA-identical donor. No insertional mutagenesis events have been observed in more than 40 ADA-SCID patients treated so far in the context of different clinical trials worldwide, suggesting a favorable risk-benefit ratio for this disease. On the other hand, the occurrence of insertional oncogenesis in SCID-X1, WAS, and chronic granulomatous disease (CGD) RV clinical trials prompted the development of safer vector construct based on self-inactivating (SIN) retroviral or lentiviral vectors (LVs). Here we present the recent results of LV-mediated gene therapy for WAS showing stable multilineage engraftment leading to hematological and immunological improvement, and discuss the differences with respect to the WAS RV trial. We also describe recent clinical results of SCID-X1 gene therapy with SIN γ-RV and the perspectives of targeted genome editing techniques, following early preclinical studies showing promising results in terms of specificity of gene correction. Finally, we provide an overview of the gene therapy approaches for other PIDs and discuss its prospects in relation to the evolving arena of allogeneic transplant.