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Some gene therapy strategies are compromised by the levels of gene expression required for therapeutic benefit, and also by the breadth of cell types that require correction. We designed a lentiviral vector system in which a transgene is under the transcriptional control of the short form of constitutively acting elongation factor 1α promoter (EFS) combined with essential elements of the locus control region of the β-globin gene (β-LCR). We show that the β-LCR can upregulate EFS activity specifically in erythroid cells but does not alter EFS activity in myeloid or lymphoid cells. Experiments using the green fluorescent protein (GFP) reporter or the human adenosine deaminase (ADA) gene demonstrate 3–7 times upregulation in vitro but >20 times erythroid-specific upregulation in vivo, the effects of which were sustained for 1 year. The addition of the β-LCR did not alter the mutagenic potential of the vector in in vitro mutagenesis (IM) assays although microarray analysis showed that the β-LCR upregulates ~9% of neighboring genes. This vector design therefore combines the benefits of multilineage gene expression with high-level erythroid expression, and has considerable potential for correction of multisystem diseases including certain lysosomal storage diseases through a hematopoietic stem cell (HSC) gene therapy approach.

Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.

Lentiviral vectors (LVs) are highly attractive as a gene therapy agent as they are able to stably integrate their genomes in both dividing and nondividing cells and, in principle, provide long-term therapeutic benefit. However, their performance in skeletal muscle in adult animals has, to date, been disappointing. In order to gain clearer insight into their utility in this tissue type, we have conducted an extensive quantitative comparison of constitutive and muscle-specific promoter activities in skeletal muscle and nonmuscle systems following LV delivery in cell lines and neonatal mice. Our data show that LV delivery to hind leg skeletal muscle of neonatal mouse results in long-term transgene expression in adulthood. We find that the human desmin (DES) promoter/enhancer is the first muscle-specific control region to match the activity of the highly active constitutive human cytomegalovirus (hCMV) promoter/enhancer in skeletal muscle within a LV context both in vitro and in vivo. Furthermore, the DES promoter/enhancer provides six- to eightfold greater expression per viral copy than the muscle-specific human muscle creatine kinase (CKM) promoter/enhancer. DES also confers a more reproducible and tissue-specific transgene expression profile compared to CKM and is therefore a highly attractive regulatory element for use in muscle gene therapy vectors.

Sickle cell disease (SCD, OMIM 603903) is one the most common inherited blood disorders worldwide, caused by a single amino acid substitution (E6V) in the β-globin chain. SCD is associated with abnormal red cell morphology, hemolytic anemia, vascular occlusion, severe pain, progressive organ damage and reduced life expectancy. Gene therapy by transplantation of autologous, genetically corrected hematopoietic stem cells could be a therapeutic alternative, particularly in patients lacking an allogeneic bone marrow donor. Recent attempts to treat β-thalassemia and SCD with gene therapy showed encouraging results as well as potential limits of a technology based on complex lentiviral vectors for stem cell transduction due to the vector size and complexity, and limited globin synthesis. Designing an efficient vector that combines high-level globin expression with high titer and infectivity remains a formidable challenge. The GLOBE vector carries a β-globin gene under the control of its promoter and a reduced version of the β-globin locus control region (LCR); it was designed to reduce the size and complexity of the LCR while maintaining its enhancer and chromatin opening activity (Miccio et al., PNAS 2008). Based on the GLOBE design, we developed novel LVs for SCD gene therapy, carrying a modified transgene encoding an anti-sickling β-globin gene with 3 amino acid substitutions (AS3), alternative β-globin promoters and a short sequence (FB) with insulating and enhancer-blocking activity in the LTR. The vectors are produced at high titer and reproducibly transduce 60-70% of human BM CD34+ cell-derived clonogenic progenitors at an average VCN of 3. The GLOBE vectors were compared to the Lenti/βAS3-FB vector (Romero, Urbinati et al. JCI 2013), previously developed for a gene therapy clinical trial of SCD. We will present data comparing the different vectors for transduction efficiency in BM CD34+ cells from healthy donors and SCD patients, βAS3-globin transgene expression and anti-sickling activity, in vivo repopulation activity in NSG mice and vector integration profiles in repopulating cells before and after transplantation. This study provides a comprehensive analysis of different LV vectors, useful to determine the optimal candidate for gene therapy of SCD that can provide high transduction of Hematopoietic Stem and Progenitor Cells (HSPCs) as well as maintaining an adequate expression of the anti-sickling globin gene.

Thalassemia is amongst the commonest single-gene disorders worldwide, caused by deficient production of α- or β-globin. Of particular clinical relevance is β-thalassemia, which as a severe monogenic disease of the hematopoietic system is an ideal target for gene therapy, either by gene addition or gene correction. Problems inherent to the gene-augmentation approach for β-thalassemia, including insertional mutagenesis or low expression of the therapeutic transgene, may be avoided using targeted gene correction of mutations with site-specific designer nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALENs). Our study is focused on the development of a personalized gene-correction therapy of the common β-thalassemia mutation, HBBIVS1-110, which in most Mediterranean and many Western countries has a frequency of above 20% (with 80% on the island of Cyprus) amongst β-thalassemia carriers. HBBIVS1-110 creates an abnormal splice-acceptor site in intron I of the β-globin gene, leading to a pre-mature in-frame stop codon in the aberrantly spliced mRNA and to early transfusion dependence of homozygotes. We have developed and evaluated designer nucleases targeting the site of the HBBIVS1-110 β-thalassemia mutation, including direct comparison of targeted disruption efficiency and toxicity for CRISPR/Cas9 and TALEN tools in human embryonic kidney cells and in murine erythroleukemia (MEL) cells carrying a HBBIVS1-110mutant transgene based on the GLOBE (MA821) vector. Using the latter, we have assessed the therapeutic efficiency and cleavage properties of these designer nucleases. Analyses included immunoblots, absolute quantification of correctly and aberrantly spliced HBB mRNAs using multiplex RT-qPCR, sequencing and characterization of the resulting genome-editing events. We noted superior performance of several TALEN pairs compared to a single CRISPR/Cas9 nuclease suitable for the target site. Moreover, we demonstrate a significant increase of correct splicing and β-globin chain synthesis for genome-edited transgenic MEL MA821 HBBIVS1-110 cells. For the two most efficient TALEN pairs, β-globin protein levels in pools of edited cells reach over 20% of that detected in MEL cells harbouring a wild-type MA821 HBB control, up from 1% for the mock-treated MA821 HBBIVS1-110 control. Our findings validate our approach and indicate its suitability also for the correction of other intronic disease-causing mutations.

Gene therapy for beta-thalassemia requires stable transfer of the beta-globin gene into hematopoietic stem cells (HSCs) and high and regulated Hb expression in the erythroblastic progeny. We developed a novel high-titer LV to express therapeutic level of Hb in one of the most severe murine model of beta-thalassemia. We cloned the human beta globin gene under the control of a minimal promoter and 2 elements of the LCR (HS2 and HS3), in the context of a SIN-18 LV. We evaluated the efficiency of this LV to correct the thalassemic phenotype in the th3/+ mouse mutant, that exhibit chronic anemia, anomalies in red cell size and shape, splenomegaly, extramedullary hematopoiesis (EMH) and iron accumulation in liver and spleen. The beta globin transgene expression was analyzed in the differentiated progeny of transduced HSCs, after transplantation into lethally irradiated thalassemic mice. Transplantation of LV- transduced HSCs lead to a high proportion (79%|[ndash]|100%) of donor RBCs expressing human beta globin, which remained stable up to 9 months after transplantation. HPLC analysis allowed the detection of the human beta globin chain, accounting for up to 43% of endogenous murine beta globin chains. The hematocrit, red blood cell count and hemoglobin level were markedly improved (HCT 44.90|[plusmn]|6.10; RBC 9.15|[plusmn]|1.22, HGB 12.07|[plusmn]|1.75) in comparison to the hematological parameters in control mice transplanted mock- transduced th3/+ cells (HCT 29.33|[plusmn]|2.08; RBC 5.79|[plusmn]|0.45, HGB 8.03|[plusmn]|1.12). The vector derived beta-globin synthesis was able to strongly reduced abnormalities in RBC morphology in mice transplanted with LV-transduced cells. FACS analysis of the erythroid TER119+ bone marrow subpopulations showed the restoration of normal erythroblastic maturation in the gene therapy- treated mice. Finally, the histopathological analysis demonstrated the correction of splenomegaly, EMH and drastic reduction of iron deposition in the spleen and liver. Quantitative PCR analysis revealed that the average proviral copy number ranged from 0.3 to 2.2, showing a direct correlation with the degree of phenotype correction. Analysis of single erythroid CFU-S, derived from secondary recipients, confirmed that the level of vector-derived human beta-globin was copy number dependent. Moreover, Southern Blot analysis of DNA extracted from transduced bone marrow samples indicated that the engraftment of a relatively small number of transduced HSCs was sufficient to fully correct the thalassemic phenotype in the th3/+ model. Finally, we demonstrated that LV-derived human beta globin expression was persistent and rescued the thalassemic phenotype in secondary transplanted th3/+ mice. Experiments are ongoing to test the therapeutic potential of the described LV to correct thalassemia major.

Gene therapy for beta-thalassemia is based on the transplantation of genetically-modified autologous hematopoietic stem cells (HSC) into patients affected by the severe form of disease. The genetic treatment of the hemoglobinopathies poses the general challenge of efficient level of gene transfer into HSC and high and persistent transgene expression, in the differentiated progeny of a genetically modified stem cell. The validation of a gene therapy approach to thalassemia requires to obtain results of gene correction in a broad number of patients’ cells, since different molecular defects in the beta-globin gene lead to the clinical phenotype. The heterogeneity in the molecular defects and in the proportion of alpha and non-alpha (beta, gamma and delta) chains will represent a key element to set a threshold in the amount of vector-derived beta-chain required to correct a thalassemic phenotype. Additionally, the impact of some biological parameters, such as the degree of BM erythroid hyperplasia, the BM subpopulations proportion and the apoptotic index, on the successful correction of thalassemic phenotype needs to be studied in the perspective of clinical translation. In order to address these issues, we collected samples from BM aspirates and isolated CD34+ cells from 25 beta+ and beta0 thalassemic patients, characterized by different genotypes and biochemical profiles of globin chains synthesis. A novel, erythroid specific LV expressing human beta-globin from a minimal promoter enhanced by only 2 LCR elements (HS2 and HS3) was used to transduce BM derived CD34+ cells at high efficiency (>80%). The efficacy of the beta-globin LV in correcting the human thalassemic phenotype was tested in an in vitro model of erythropoiesis and in the human-mouse hematological chimera. Upon transduction, normal level of HbA expression was achieved in erythroblastic cultures and BFU-E, associated with a progression towards erythroid maturation, which was impaired in mock-transduced thalassemic cells. Molecular analysis showed proviral integrity, with no detectable rearrangements and an average proviral copy number of 2.4. Analysis of specific globin chains proportion and contribution to phenotype correction in the context of different genotypes is under evaluation.