Gene Therapy For Inherited Blood Diseases, From Viral Vectors To Gene Editing

Twenty-five years ago, genetically modified bone marrow cells were administered for the first time to a child suffering from adenosine deaminase (ADA) deficiency, a rare disorder of the immune system. Since then, gene therapy has struggled to find its place in clinical medicine, amid a rollercoaster of successes and setbacks and hype and skepticism with little precedent in modern times. Recently, a series of authoritative clinical studies proved that transplantation of genetically modified hematopoietic stem cells can cure severe diseases like immunodeficiencies, hemoglobinopathies and metabolic diseases, contributing to transforming gene therapy into one of the hottest area of investment for the biotechnology and the pharma industry. The basic technology for the genetic modification of stem cells relies on retroviral vectors, and particularly on those derived from oncoretroviruses or lentiviruses, such as HIV-1. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. Addressing these issues brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. More recently, a new generation of technology emerged, aimed at correcting the genome rather than replacing defective gene function. This technology relies on designer nucleases capable of generating double- or single-stranded breaks in genomic DNA, which are then repaired either by error-prone non homologous end-joining or by the more precise homologous recombination. This allows generating knock-out mutations or repairing genes with remarkable precision and efficiency in many cell types. At Genethon, we are using lentiviral vector technology to correct Wiskott-Aldrich syndrome, X-linked SCID, chronic granulomatous disease and sickle-cell disease, while developing CRISPR/Cas9-based genome editing for a number of applications

131. Targeted Genome Editing in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the survival-motor-neuron 1 (SMN1) telomeric gene. Deficiencies in the ubiquitous SMN function affect multiple tissues and organs; however neuronal tissue is primarily sensitive, resulting in α-motor neuron degeneration in the ventral horn of the spinal cord with subsequent neuromuscular-junction dysfunction and proximal muscle weakness. The onset of disease and degree of severity are variable in patients and they are determined in part by multiple copies of the centromeric homologue SMN2 that inversely correlate with the phenotypic severity. Indeed, SMN2 gene mainly produces a truncated form SMNΔ7 by aberrant alternative splicing and a small amount (~10%) of the fully active full-length SMN, thus buffering the SMN deficiency. A potential strategy for treating SMA patients is to increase SMN levels in the affected tissues, hence gene therapy and modifiers of SMN2-alternative splicing have proved therapeutic efficacy in SMA animal models.In this study, we explored the possibility of applying targeted genome editing technology to the human SMN locus in order to revert the SMN2 sequence to a SMN1-like sequence that may undergo proper splicing under the the endogenous transcriptional control. The resulting correction would be permanent and lead to longlasting protein production in gene-edited cells. We used the streptococcus pyogenes Cas9-CRISPR system to target the SMN2 gene at different locations. Two main strategies were explored: i) SMN1_exon7 addition/correction by promoting homology-driven DNA repair, ii) SMN2_intron7_ intronic-splicing-silencer (ISS-N1)mutation and correction of SMN2 aberrant splicing, by exploiting the non-homologous end-joining (NHEJ) pathway. Plasmids encoding Cas9-GFP under the control of CMV promoter, and selected gRNAs downstream to the Pol-III U6 promoter (Addgene) were transfected in HEK-293T cell line and in immortalized myoblasts derived from either healthy donors or SMA patients. Transfection efficiency was estimated as percentage of GFP-expressing cells (20-50% and 1-10%, respectively) and nuclease activity detected by Surveyor assay and target site sequencing. In particular, in SMA patient-derived myoblasts we detected mutations (indels) at the level of the induced DNA double-strand break at ~30% frequency. Levels of SMN restoration will be investigated by qPCR of the different species of SMN transcripts and by western blotting of SMN protein. The goal of this study is to provide an in vitro proof of principle of effective gene correction in SMA patient-derived cells. In the context of a multisystemic, complicated disease such as SMA, targeted genome editing strategy could represent an additional therapeutic tool

Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivo by the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications

T Lymphocytes Transduced with a Lentiviral Vector Expressing F12-vif Are Protected from HIV-1 Infection in an APOBEC3G-Independent Manner

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function

606. Identification of a 45-aa Domain of the F12-Vif Mutant Possessing Anti-HIV Activity

Our previous results have demonstrated that T-cell lines and primary T lymphocytes transduced with a Tat-dependent HIV-based lentiviral vectors expressing the mutant isoform of the vif gene, F12-vif, are protected from HIV-1 infection. F12-Vif is a 192-aa natural variant polypeptide owing 14 unique amino acid substitutions. The substitutions are randomly scattered along the entire sequence with the exception of a 5-aa cluster located at positions 127, 128, and 130|[ndash]|132. None of the 14 aa substitutions is present in the SOCS box that recruits the E3 ubiquitin ligase responsible of APOBEC3G (AP3G) degradation during HIV infection. In line with this notion, we have shown that the antiviral function of F12-Vif is not due to a dominant negative feature of the mutant in regards to the Vif-mediated degradation of AP3G rather to some other unknown means. Therefore, in the effort to elucidate the F12-Vif mechanism of action, we started to identify the protein domain of F12-Vif responsible of HIV-1 inhibition. To this end, we have constructed three chimeric genes (Chim1, Chim2 and Chim3) composed by wild-type and F12-vif regions. T cell lines and cord blood derived CD4+T lymphocytes were transduced with the lentiviral vectors expressing the chimeric genes and then challenged with both X4 and R5 HIV-1 strains. We show that 45 amino acids in the C-terminal domain of the F12-Vif mutant are sufficient to exert anti-viral effect in transduced cells. In contrast to F12-Vif, Chim3 does not allow the rescue of the replication of a vif-deficient HIV-1 in the context of either X4 or R5 tropism in non permissive cells. This specific feature renders Chim3 a truly dominant negative protein more suitable than F12-Vif for an anti-HIV gene therapy approach

The GATA1-HS2 Enhancer Allows Persistent and Position-Independent Expression of a β-globin Transgene

Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing

C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein δ (C/EBPδ), Bmi1, and ΔNp63α identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPδ and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPδ inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27Kip1 and p57Kip2. These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBPδ, but not ΔNp63α, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBPδ is recruited to the chromatin of positively (p27Kip1 and p57Kip2) and negatively (p16INK4A and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity

811. Correction of Laminin-5 β3 Chain Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Mutations in any of the genes encoding the laminin 5 heterotrimer (|[alpha]|3, |[beta]|3 and |[gamma]|2) cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. We and others have shown that expression of a retrovirally transferred |[beta]|3-chain cDNA in keratinocytes from affected patients reconstitutes normal synthesis, assembly and secretion of laminin 5, and corrects the adhesion defect in vitro and in vivo. We have recently started a phase-I clinical trial of gene therapy of JEB based on transplantation of cultured skin derived from autologous epidermal stem cells transduced with a MLV-derived retroviral vector. Since gamma- retroviral vectors have raised safety concerns for the genotoxic risk associated with the insertion of LTR elements into the human genome, we developed an alternative gene transfer strategy based on LTR- modified, HIV-derived lentiviral vectors. Two self-inactivating (SIN) lentiviral vectors were built, in which expression of either GFP or a LAMB3 cDNA is under the control of either a constitutive promoter (PGK) or the keratinocyte-specific, 2.2-kb promoter-enhancer of keratin 14 (K14). In a third construct, expression of the transgene is under the control of the viral LTR, modified by replacing the U3 region with two K14 enhancer elements. Analysis in human keratinocyte cultures and in full-thickness human skin equivalents reconstituted onto immunodeficient mice showed that GFP expression directed by the K14 elements is tissue-specific and restricted to the basal layer of the epidermis. Expression of laminin5 from the three alternative vectors was evaluated in keratinocyte cultures derived from skin biopsies of JEB patients. Biochemical and cell kinetics assays demonstrated transduction of epidermal clonogenic stem/progenitor cells and full phenotypic correction of JEB keratinocytes with all vectors. Southern blot analysis of individual cell clones showed that LTR-modified lentiviral vectors are genetically stable and integrate in multiple copies in the human genome. This study shows that the use of lentiviral vectors transcriptionally targeted to the basal keratinocytes by the insertion of restricted enhancer elements is an effective, and potentially safer, alternative for gene therapy of JEB

826. Transduction of Human Hematopoietic Stem Cells by RD114-TR-Pseudotyped Lentiviral Vectors

HIV-1-derived lentiviral vectors are efficiently pseudotyped by a chimeric envelope (RD114-TR) encoding the extracellular and transmembrane domains of the FLV RD114 glycoprotein fused to cytoplasmic tail (TR) of the MLV 4070A amphotropic glycoprotein. RD114-TR pseudotyped vectors may be concentrated by centrifugation, are resistant to complement inactivation, and are of particular interest for both ex vivo and in vivo gene therapy applications. We carried out a comparative analysis of VSV-G and RD114-TR-pseudotyped lentiviral vectors in transducing human cord blood-derived CD34+ hematopoietic stem/progenitor cells. Transduction efficiency was comparatively analysed in CD34+ cells in liquid culture, in the progeny of CD34+ clonogenic progenitors in semi-solid culture, and in the progeny of CD34+ repopulating stem cells after xeno-transplantation in NOD-SCID mice. In all cases, RD114-TR-pseudotyped vectors transduced hematopoietic cells at lower m.o.i., resulting in lower toxicity and more efficient stable transduction at comparable vector copy number per genome. Potential changes in CD34+ cells transcription profile and phenotype upon transduction with RD114-TR or VSV-G-pseudotyped vectors was investigated by Affymetrix Gene Chips microarray analysis. We found no significant difference in gene expression patterns between mock-RD114-TR and VSV-G-transduced cells. Our study show that the biology of repopulating hematopoietic stem cells and their progeny is not affected by transduction with RD114-TR-pseudotyped lentiviral vectors

Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity \u2018\u2018sandwich\u2019\u2019 (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy application