Influence of insulators on transgene expression from integrating and non-integrating lentiviral vectors.

International audienceBACKGROUND: The efficacy and biosafety of lentiviral gene transfer is influenced by the design of the vector. To this end, properties of lentiviral vectors can be modified by using cis-acting elements such as the modification of the U3 region of the LTR, the incorporation of the central flap (cPPT-CTS) element, or post-transcriptional regulatory elements such as the woodchuck post-transcriptional regulatory element (WPRE). Recently, several studies evaluated the influence of the incorporation of insulators into the integrating lentiviral vector genome on transgene expression level and position effects. METHODS: In the present study, the influence of the matrix attachment region (MAR) of the mouse immunoglobulin-κ (Ig-κ) or the chicken lysozyme (ChL) gene was studied on three types of HIV-1-derived lentiviral vectors: self-inactivating (SIN) lentiviral vectors (LV), double-copy lentiviral vectors (DC) and non-integrating lentiviral vectors (NILVs) in different cell types: HeLa, HEK293T, NIH-3T3, Raji, and T Jurkat cell lines and primary neural progenitors. RESULTS AND DISCUSSION: Our results demonstrate that the Ig-κ MAR in the context of LV slightly increases transduction efficiency only in Hela, NIH-3T3 and Jurkat cells. In the context of double-copy lentiviral vectors, the Ig-κ MAR has no effect or even negatively influences transduction efficiency. In the same way, in the context of non-integrating lentiviral vectors, the Ig-κ MAR has no effect or even negatively influences transduction efficiency, except in differentiated primary neural progenitor cells.The ChL MAR in the context of integrating and non-integrating lentiviral vectors shows no effect or a decrease of transgene expression in all tested conditions. CONCLUSIONS: This study demonstrates that MAR sequences not necessarily increase transgene expression and that the effect of these sequences is probably context dependent and/or vector dependent. Thus, this study highlights the importance to consider a MAR sequence in a given context. Moreover, other recent reports pointed out the potential effects of random integration of insulators on the expression level of endogenous genes. Taken together, these results show that the use of an insulator in a vector for gene therapy must be well assessed in the particular therapeutic context that it will be used for, and must be balanced with its potential genotoxic effects

Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques

The authors describe quantitatively and qualitatively different forms of experimental autoimmune encephalitis (EAE) in cynomolgus macaques. They found that bacterial contaminants within recombinant human myelin oligodendrocyte glycoprotein seemed to aggravate disease evolution. They provide anatomopathological features of fulminant and progressive forms of EAE, allowing them to distinguish specific factors influencing the evolution of this model of autoimmune demyelinating disease. Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression

Grafted Human Embryonic Progenitors Expressing Neurogenin-2 Stimulate Axonal Sprouting and Improve Motor Recovery after Severe Spinal Cord Injury

7 p.Background: Spinal cord injury (SCI) is a widely spread pathology with currently no effective treatment for any symptom. Regenerative medicine through cell transplantation is a very attractive strategy and may be used in different non-exclusive ways to promote functional recovery. We investigated functional and structural outcomes after grafting human embryonic neural progenitors (hENPs) in spinal cord-lesioned rats.Methods and Principal Findings: With the objective of translation to clinics we have chosen a paradigm of delayed grafting, i.e., one week after lesion, in a severe model of spinal cord compression in adult rats. hENPs were either naive or engineered to express Neurogenin 2 (Ngn2). Moreover, we have compared integrating and non-integrating lentiviral vectors, since the latter present reduced risks of insertional mutagenesis. We show that transplantation of hENPs transduced to express Ngn2 fully restore weight support and improve functional motor recovery after severe spinal cord compression at thoracic level. This was correlated with partial restoration of serotonin innervations at lumbar level, and translocation of 5HT1A receptors to the plasma membrane of motoneurons. Since hENPs were not detectable 4 weeks after grafting, transitory expression of Ngn2 appears sufficient to achieve motor recovery and to permit axonal regeneration. Importantly, we also demonstrate that transplantation of naive hENPs is detrimental to functional recovery.Conclusions and Significance: Transplantation and short-term survival of Ngn2-expressing hENPs restore weight support after SCI and partially restore serotonin fibers density and 5HT1A receptor pattern caudal to the lesion. Moreover, grafting of naive-hENPs was found to worsen the outcome versus injured only animals, thus pointing to the possible detrimental effect of stem cell-based therapy per se in SCI. This is of major importance given the increasing number of clinical trials involving cell grafting developed for SCI patients.This study was supported by the European Union FP6 "RESCUE" STREP; the "Institut pour la Recherche sur la Moelle Epiniere"; the "Academie de Medecine"; the "Societe Francaise de Neurochirurgie"; "Verticale" and the "Association Demain Debout Aquitaine". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Non-human primates are essential models in the translational research of multiple sclerosis

Ageing Western societies are facing an increasing prevalence of chronic inflammatory and degenerative diseases for which no effective treatments exist. The pressure on the drug development industry to develop such treatments creates a need for translationally relevant animal models, which faithfully replicate essential pathogenic mechanisms of the human disease. In this Short Review, we discuss the essential role of the non-human primate (NHP) in the translational research into the pathogenesis and treatment of the autoimmune neurological disease multiple sclerosis (MS)

Integration of genetically modified adult astrocytes into the lesioned rat spinal cord

Combination of ex vivo gene transfer and cell transplantation is now considered as a potentially useful strategy for the treatment of spinal cord injury. In a perspective of clinical application, autologous transplantation could be an option of choice. We analyzed the fate of adult rat cortical astrocytes genetically engineered with a lentiviral vector transplanted into a lesioned rat spinal cord. Cultures of adult rat cortical astrocytes were infected with an HIV-1-derived vector (TRIP-CMV-GFP) and labeled with the fluorescent dye Hoechst. Transfected and labeled astrocyte suspension was injected at T11 in rats in which spinal cord transection at T7-T8 levels had been carried out 1 week earlier. Six weeks after grafting, the animals were sacrificed and transplants were retrieved either by Hoechst fluorescence or by immunohistochemistry for detection of glial fibrillary acidic protein (GFAP) and vimentin. Grafted astrocytes expressing green fluorescent protein (GFP) were found both at the injection and transection sites. Genetically modified astrocytes thus survived, integrated, and migrated within the host parenchyma when grafted into the completely transected rat spinal cord. In addition, they retained some ability to express the GFP transgene for at least 6 weeks after transplantation. Adult astrocytes infected with lentiviral vectors can therefore be a valuable tool for the delivery of therapeutic factors into the lesioned spinal cord.Contract grant sponsor: Institut de Recherche sur la Moelle Epiniere;Contract grant sponsor: Association Francaise des Myopathies.Peer reviewe

Cranial nerve involvement in patients with MOG antibody–associated disease

International audienc

Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable

Both Systemic and Intra-articular Immunization with Citrullinated Peptides Are Needed to Induce Arthritis in the Macaque

ObjectivesAnti-citrullinated peptides antibodies (ACPAs) have high specificity for the diagnosis of rheumatoid arthritis (RA), but their role in the pathophysiology is not fully established. The main genetic risk factor for RA, the shared epitope in major histocompatibility complex class II, is associated with ACPAs. Among certain non-human primates, 8% carry the shared epitope called H6 haplotype, and being similar to humans, are ideal candidates to study the role of ACPAs in RA. The goal of this study was to develop a macaque model of RA based on immunization against citrullinated peptides to generate an ACPA-mediated model of arthritis.MethodsCynomolgus macaques were immunized with four citrullinated peptides from vimentin, fibrinogen, and aggrecan, known to induce T-cell response in RA patients, and received an intra-articular (IA) boost with the same four citrullinated peptides pooled.ResultsIn the macaque, the T-cell response was specific to citrullinated peptides. Antibodies generated in response to immunization were cross-reactive between the citrulline and arginine peptides. The presence of the H6 haplotype did not affect the magnitude of the immune response. Since no clinical response was observed, macaques received an IA boost with the same four peptides pooled and incomplete Freund’s adjuvant, which led to a prolonged neutrophil-rich mono-arthritis, preferentially in H6-positive animals. Conversely, animals boosted with incomplete Freund’s adjuvant alone presented only transient mono-arthritis.ConclusionThis two-hit model of prolonged mono-arthritis mimics what could happen in RA. Despite the limited number of joints with disease in the macaque model, the model appears unique to study the events occurring during the preclinical phase of RA, from immunization against citrullinated peptides to the clinical appearance of disease