Academic literature on the topic 'Human herpes virus; Gene therapy; DNA'

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Background The use of opioids to treat pain is often limited by side effects mediated through the central nervous system. The current study used a recombinant herpes simplex virus type 1 to increase expression of the mu-opioid receptor (muOR) in primary afferent neurons. The goal of this strategy was to enhance peripheral opioid analgesia. Methods Cutaneous inoculation with herpes simplex virus containing muOR complementary DNA (cDNA) in antisense (SGAMOR) or sense (SGMOR) orientation relative to a constitutive promoter, or complementary DNA for Escherichia coli lac Z gene as a control virus (SGZ) was used to modify the levels of muOR in primary afferents. The effects of altered muOR levels on peripheral analgesia were then examined. Results At 4 weeks after SGAMOR and SGMOR infection, decreased and increased muOR immunoreactivity was observed in ipsilateral dorsal hind paw skin, lumbar dorsal root ganglion cells, and superficial dorsal horns, respectively, compared with SGZ. This change in muOR expression in mice by SGAMOR and SGMOR was accompanied at the behavioral level with a rightward and leftward shift in the loperamide dose-response curve, respectively, compared with SGZ. Conclusions This gene therapy approach may provide an innovative strategy to enhance peripheral opioid analgesia for the treatment of pain in humans, thereby minimizing centrally mediated opioid side effects such as sedation and addiction.

Infection with herpes simplex virus (HSV) types 1 and 2 is ubiquitous in the human population. Most commonly, virus replication is limited to the epithelia and establishes latency in enervating sensory neurons, reactivating periodically to produce localized recurrent lesions. However, these viruses can also cause severe disease such as recurrent keratitis leading potentially to blindness, as well as encephalitis, and systemic disease in neonates and immunocompromised patients. Although antiviral therapy has allowed continual and substantial improvement in the management of both primary and recurrent infections, resistance to currently available drugs and long-term toxicity pose a current and future threat that should be addressed through the development of new antiviral compounds directed against new targets. The development of several promising HSV vaccines has been terminated recently because of modest or controversial therapeutic effects in humans. Nevertheless, several exciting vaccine candidates remain in the pipeline and are effective in animal models; these must also be tested in humans for sufficient therapeutic effects to warrant continued development. Approaches using compounds that modulate the chromatin state of the viral genome to suppress infection and reactivation or induce enhanced antiviral immunity have potential. In addition, technologies such as CRISPR/Cas9 have the potential to edit latent viral DNA in sensory neurons, potentially curing the neuron and patient of latent infection. It is hoped that development on all three fronts—antivirals, vaccines, and gene editing—will lead to substantially less HSV morbidity in the future.

The majority of viral vectors currently used possess modest cargo capability (up to 40 kb) being based on retroviruses, lentiviruses, adenoviruses, and adenoassociated viruses. These vectors have made the most rapid transition from laboratory to clinic because their small genomes have simplified their characterization and modification. However, there is now an increasing need both in research and therapy to complement this repertoire with larger capacity vectors able to deliver multiple transgenes or to encode complex regulatory regions, constructs which can easily span more than 100 kb. Herpes Simplex Virus Type I (HSV-1) is a well-characterized human virus which is able to package about 150 kb of DNA, and several vector systems are currently in development for gene transfer applications, particularly in neurons where other systems have low efficiency. However, to reach the same level of versatility and ease of use as that of smaller genome viral vectors, simple systems for high-titer production must be developed. This paper reviews the major HSV-1 vector systems and analyses the common elements which may be most important to manipulate to achieve this goal.

Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancers. In spite of initial good response to chemotherapy, the prognosis of TNBC remains poor and no effective specific targeted therapy is readily available. Recently, we demonstrated the ability of U94, the latency gene of human herpes virus 6 (HHV-6), to interfere with proliferation and with crucial steps of the metastatic cascade by using MDA-MB 231 TNBC breast cancer cell line. U94 expression was also associated with a partial mesenchymal-to-epithelial transition (MET) of cells, which displayed a less aggressive phenotype. In this study, we show the ability of U94 to exert its anticancer activity on three different TNBC cell lines by inhibiting DNA damage repair genes, cell cycle and eventually leading to cell death following activation of the intrinsic apoptotic pathway. Interestingly, we found that U94 acted synergistically with DNA-damaging drugs. Overall, we provide evidence that U94 is able to combat tumor cells with different mechanisms, thus attesting for the great potential of this molecule as a multi-target drug in cancer therapy.

The paper describes the influence of selected genes on neogenesis, including their expression, RNA transformation, translation and transcription. The role of adhesive molecules, extracellular matrix, cytoskeleton and signal conduction in neoplastic induction is described. External stimuli, internal factors and disturbances in DNA repair can cause cell mutations (fig. 1). An accumulation of various factors in different gene classes, together with their amplification, leads to tumour formation. Neoplastic cells undergo a dominant mutation, thereby gaining a new function, or cumulate recessive mutations which cause the loss of a function. This is particularly true in genetic anomalies associated with the cadherin system, e.g. the loss of E-cadherin expression in mammary cancer. The loss of E-cadherin or catenin expression causes the loss of cell connections, which facilitates metastasizing. Cells in metastases often show genetic disorders, a more malignant phenotype and increased drug resistance, which worsens clinical prognosis. The search for new anti-neoplastic drugs for humans is based on molecular studies and mice experimental models. The animals in these models show a phenotype corresponding with specific human diseases, e.g. Pax gene mutation in sarcomas and carcinomas, antisense DNA therapy (in Burkitt’s lymphoma or chronic leukaemia) or induction of retroviral vectors (thymidine kinase gene) in herpes virus (HS-th) in proliferating cells in multiform glioblastoma.

ABSTRACT Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep − HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep + HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep − vector 3′ AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep +, but not the rep −, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep + hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.

Efficient and specific delivery of nucleic acid (NA) therapeutics to tumor cells is extremely important for cancer gene therapy. Various therapeutic strategies include delivery of DNA-therapeutics such as immunostimulatory or suicide genes and delivery of siRNA-therapeutics able to silence expression of cancer-related genes. Peptides are a promising class of non-viral vehicles which are biodegradable and can efficiently condense, protect and specifically deliver NA to the cells. Here we designed arginine-histidine-rich peptide carriers consisting of an iRGD ligand to target αvβ3 integrins and studied them as vehicles for DNA and siRNA delivery to cancer cells. Combination of iRGD-modified and unmodified arginine–histidine-rich peptides during NA complexation resulted in carriers with different ligand contents. The NA-binding and protecting properties in vitro transfection efficiency and cytotoxicity of the DNA- and siRNA-polyplexes were studied and the most efficient carrier RGD1 was determined. The ability of the peptides to mediate specific intracellular uptake was confirmed inhuman cervical carcinoma (HeLa), human kidney (293T) and human pancreatic (PANC-1) cell lines with different αvβ3 integrins surface expression. By means of RGD1 carrier, efficient delivery of the herpes simplex virus (HSV-1) thymidine kinase gene to PANC-1 cells was demonstrated. Subsequent ganciclovir treatment led to a reduction of PANC-1 cells’ viability by up to 54%. Efficient RNAi-mediated down-regulation of GFP and VEGFA gene expression was achieved in MDA-MB-231-GFP+ breast cancer and EA.hy926 endothelial cells, respectively, by means of RGD1/siRNA polyplexes. Here we demonstrated that the peptide carrier RGD1 can be considered as promising candidate for development of NA therapeutics delivery systems useful in cancer gene therapy.

Abstract Abstract 4753 Introduction Plasmablastic lymphoma (PBL) is an aggressive and generally fatal, rare type of B-cell non-Hodgkin's lymphoma (NHL) with predilection to sino-oral mucosa. PBL is usually associated with human immunodeficiency virus (HIV) and displays an unusual phenotype lacking the expression of B-cell surface antigens. PBLs are infected by Epstein Barr virus (EBV) in approximately 80% of cases and exhibit a restricted pattern of EBV gene expression as they do not typically express latent membrane proteins (LMPs), which enforce viral latency. Azidothymidine (AZT), a thymidine analogue, is an excellent substrate for EBV thymidine kinase, and is capable of inducing EBV lytic gene expression and apoptosis in primary Type I latency EBV+ Burkitt lymphoma (BL) cell lines. AZT was initially developed as an antineoplastic agent but was found to have low efficacy due to poor affinity to human DNA polymerase and low incorporation into DNA. The chemotherapy drug methotrexate (MTX), which also induces gamma-herpes virus lytic induction, inhibits thymidylate synthase thus blocking de novo synthesis of dTMP increasing the likelihood of AZT incorporation into DNA. Indeed, the combination of high-dose MTX and AZT was found to be clinically efficacious in HIV-infected patients with aggressive relapsed NHL. Similarly, at the University of Miami, we have found this drug combination to be highly effective when used in patients with aggressive gamma-herpes virus lymphomas including EBV+ BL and human herpes virus 8 (HHV-8) related primary effusion lymphoma (solid PEL). We report here the role of high dose MTX plus intravenous AZT with alternating infusional EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and adriamycin) in the treatment of HIV associated EBV+ plasmablastic lymphoma. Methods Three HIV+ patients with biopsy proven, EBV-encoded RNA (EBER)+ PBL in sino-oral mucosa were treated with high dose methotrexate (4.5 g/m2 IV on day 1) and AZT 1.5 g IV infusion q12 hours (days 1-3) alternating with dose-adjusted (DA) EPOCH chemotherapy for 6-8 cycles as first line therapy. Patients were started/kept on HAART and were administered prophylactic intrathecal MTX±cytarabine. Responses were evaluated by oral exam and CT scans. Results Selected characteristics of patients are demonstrated in Table. Only Patient 2 was on HAART at the time of diagnosis. Patient 1 had intracranial extension of the large left maxillary lesion through sphenoid sinus with negative CSF cytology. All patients treated with HD-MTX/AZT and alternating EPOCH as above achieved a complete remission (CR). Patient 1 achieved CR after the 3rd cycle. However, 19 days after the 4th cycle he was found to have local recurrence, which was thought to be secondary to poor chemotherapy penetration to necrotic oral palate tissue, and was administered a total of 4620 cGy intensity-modulated radiotherapy to head and neck region followed by consolidation with the same chemotherapy regimen for 4 more cycles. He remains alive and free of disease after 14 months. Patient 2 and 3 received a total of 6 cycles of chemotherapy and remain disease-free after 12 and 13 months, respectively. No grade ≥3 toxicities were encountered during the treatment. Conclusion The combination of high dose MTX and AZT plus alternating DA-EPOCH chemotherapy is a tolerable and effective treatment for HIV related EBV+ plasmablastic lymphomas. The combination of MTX and AZT is a highly active, EBV lytic inducing and targeted regimen which deserves further investigation in the treatment of gamma-herpes virus associated malignancies. Disclosures: No relevant conflicts of interest to declare.

The interplay between the endocrine and immune systems has come into focus in recent years with the insight that endocrine parameters may affect susceptibility to both auto-immune and infectious diseases. Our interest in immunoendocrine regulation led us to investigate the effects of glucocorticoids on Herpes simplex virus type 1 (HSV-1) infections. Glucocorticoids used to treat inflammatory conditions are not yet recommended for HSV-1 therapy, since they have been reported to prolong viral shedding both in vivo and in vitro. Here we report that glucocorticoids did not alter the viral yield in human gingival fibroblast (HGF) cell culture when glucocorticoid treatment and viral infection occured simultaneously, but the viral yield increased when cells were treated with the glucocorticoid dexamethasone (dex) prior to viral infection. We found that viral infection in our primary cell system increased NF-kappaB levels and DNA binding. In addition, the amount of glucocorticoid receptor (GR) increased following viral infection, and HSV-1 infection as such could induce glucocorticoid-driven transcription of a reporter gene in human embryo kidney (HEK) 293 cells stably transfected with GR. Dex treatment did not affect HSV-1-induced binding of p65 to an NF-kappaB element in an electrophoretic mobility shift assay, and acyclovir was still efficient as an anti-viral drug in the presence of dex. Further studies of the observed effects of HSV-1 infection and glucocorticoid treatment on GR and NF-kappaB regulation could give insights into the immunoendocrine mechanisms important for defence and therapy against viral infections.

The human c-myc gene is a cellular proto-oncogene composed of three exons and two introns. Transcription of c-myc is controlled by two promoters, Pl and P2. The activity of these promoters is regulated by many factors, such as cellular transcription factors E2F, YYl, and HSV-1 immediate-early proteins, ICPO, ICP4. Many regulatory elements located both upstream of and between P 1 and P2 have been identified, and some of these are required for optimum expression of c-myc. In this thesis research, a region downstream from P2 in the c-myc exon 1 was identified by its response to transactivation by HSV-1 immediate-early proteins, ICPO and ICP4. The purpose of this research was to examine this region for regulatory sites that respond to HSV-1 infection. I hypothesized that after HSV-1 infection, ICPO and ICP4 activate c-myc expression, in part, through regulatory sequences present in exon 1. To test for this hypothesis, reporter plasmids containing (I) the c-myc promoter (from - 101 bp relative to Pl) and exon 1 coupled to the bacterial CAT gene were constructed. (ii) The c-myc exon sequences used were either intact (wild-type) or they were constructed with various deletions. The activities of these plasmids were examined in transient expression assays. To analyze protein binding, electrophoretic mobility shift assay (EMSA) and completion EMSAs were carried out. The results from these experiments lead to the following conclusions: (i) ICP4 and ICPO serve as activators, whereas ICP27 inhibits c-myc gene expression. (ii) The region from +332 to +513 within the c-myc exon 1 contains an important element required for transactivation of the c-myc gene by HSV-1 proteins. (iii) Cellular proteins, including factor YYl, bind to the region from +332 to +513 in the c-myc exon 1. Although the exact mechanism by which HSV-1 immediate-early proteins regulate cmyc gene expression is still not clear, it gives rise to a possibility that this regulation is caused by turning on or activation of the cellular regulatory proteins by ICP4 and ICPO. The cellular proteins in turn activate the c-myc gene expression by interacting with the ciselement downstream from P2.

Development of safe and efficient approaches for gene delivery in human embryonic stem cells (hESc) and particularly in human induced pluripotent stem (hiPS) cells, which can be derived in a person-specific manner, is considered to be imperative for harnessing their full potential in both the basic and applied research. The aim of this study was to evaluate the potential of human artificial chromosome (HAC) for gene delivery and expression in hESc and hiPS cells. HAC offers many potential advantages including the provision for carrying large genes with corresponding regulatory elements to obtain long-term regulated gene expression. In addition, they can replicate and segregate independently without integration into the host cell genome. To develop HAC in hiPS cells, the first part of the study was aimed at generating hiPS cells utilising the Herpes Simplex Virus (HSV)-1 amplicon system. With the use of EBNA-1/OriP retention elements incorporated into the HSV-1 amplicon vectors, hiPS cells completely free of vector and transgenes sequences were successfully derived from human embryonic fibroblasts. The hiPS cells exhibited proliferation and differentiation potential similar to that of hESc. In the second part of the study, development of HAC in hESc and hiPS cells was assessed by utilising the HSV-1 amplicon system to deliver the HAC DNA. Analysis of the hESc confirmed the presence of functional HAC which replicated the behaviour of the host chromosomes. Additionally, HAC generation did not lead to impairment in the developmental potential and pluripotency of hESc. The hiPS cells supported HAC at low frequency but DNA also integrated into the host chromosomes. The HAC system, therefore, needs further refinements to improve the frequency of HAC formation and reduce the chromosomal integration of HAC constructs in hiPS cells. Overall, these findings provide a simple and safe way of pluripotency induction and genetic modification of pluripotent stem cells using the HSV-1 amplicon system and represent an important advance towards patient specific gene and cell therapy.

Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.

Human immunodeficiency type 1 (HIV-1) is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years’ infection and as a result, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that a moderate protection is possible, which may correlate with antibody dependent cellular cytotoxicity (ADCC) activity. Previous studies in the Lu lab demonstrated that in an HIV-1 vaccine phase I trial, DP6-001, a polyvalent Env DNA prime-protein boost formulation, could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities along with multiple Fc mediated effector functions. I developed a protocol for the production and analysis of HIV-1 Env-specific human monoclonal antibodies (mAbs) isolated from these DP6-001 vaccinees. By utilizing a labeled gp120 bait to isolate Env specific B cells, paired heavy and light chain immunoglobulin (Ig) genes were cloned and allowed for the production of monoclonal antibodies with specificity for gp120. By using this protocol, 13 isolated mAbs from four DP6-001 vaccinees showed broad binding activities to gp120 proteins of diverse subtypes, both autologous and heterologous to vaccine immunogens, with mostly conformational epitopes and a few V3 and C5 specific mAbs. Equally cross-reactive Fc-mediated functional activities, including ADCC and antibody dependent cellular phagocytosis (ADCP), were present with both immune sera and isolated mAbs, confirming the induction of non-neutralizing functional antibodies by the DNA prime- protein boost vaccination. Elicitation of broadly reactive mAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV-1 vaccine design.

The development of safe and efficient gene delivery systems in pluripotent human embryonic stem cells (hESc) is essential to realising their full potential for basic and clinical research. The purpose of this study was to develop an efficient, non-integrating gene expression system in pluripotent hESc using human artificial chromosomes (HAC). Similar to endogenous chromosomes, HAC are capable of gene expression, replication and segregation during cell division. Unlike retroviral-mediated gene delivery vectors, HAC do not integrate into the host genome and can encompass large genomic regions for the delivery of multiple genes. Despite the advantages HAC offer, their use has been limited due to laborious cloning procedures and poor transfection efficiencies, and thus only studied in immortalised and tumour-derived human cell lines. In this study, the high transduction efficiency of herpes simplex virus type-1 (HSV-1) amplicons was utilised to overcome the described difficulties and delivered HAC vectors into pluripotent hESc. Analysis of stable hESc clones showed that de novo gene-expressing HAC were present at high frequencies ranging from 10-70% of metaphases analysed, without integrating into the genome. The established HAC contained an active centromere, and were stably maintained without integration or loss in the absence of selection for 90 days. Stable HAC-containing hESc clones retained their pluripotency as demonstrated by neuronal differentiation, in vitro germ layer and teratoma formation assays. HAC gene expression persisted, with some variation, post-differentiation in the various deriving cell types. This is the first report of successful de novo HAC formation in hESc for gene expression studies. These findings show potential for delivering high-capacity genomic constructs safely and efficiently into pluripotent cells for the purpose of genetic manipulation and ultimately patient-specific somatic gene therapy.

Le but de ce travail est de developper une nouvelle methode d'activation enzymatique d'oligonucleotides antisens ou anti-genes diriges contre le vih. La reduction par des reductases cellulaires d'un analogue de flavine fixe a un oligonucleotide associe a sa sequence complementaire cible du vih pourrait conduire a une degradation radicalaire tres efficace de celle-ci. Ce travail a tout d'abord consiste a preparer les premiers conjugues flavine-oligonucleotide. Afin de simplifier les problemes de synthese, differentes flavines modifiees portant une ou deux fonctions hydroxyles sur la chaine laterale ont ete preparees et se sont averees substrats de la flavine reductase. Une methode originale de couplage entre une flavine simplifiee preparee portant une fonction h-phosphonate et la fonction hydroxyle terminale 5' d'un oligonucleotide a ete mise au point et a permis de preparer les conjugues avec de bons rendements quelle que soit la sequence. Le comportement de ces conjugues en presence de leur sequence cible, simple ou double brin d'acide nucleique a ensuite ete etudie. La presence de la flavine liee a un oligonucleotide favorise la formation de l'hybride double brin avec sa sequence complementaire (retards sur gel, fluorimetrie et etude de la fusion des duplex). Un conjugue dirige contre la sequence ppt du vih-1 est capable de reconnaitre sa cible (experiences de prises d'empreintes) et son irradiation avec de la lumiere visible ou uv provoque des photocoupures directes et selectives au niveau de deux guanines, a proximite de la triple helice attendue. Cet oligonucleotide ne presente pas de toxicite et inhibe l'expression virale de facon significative. Les premiers resultats d'activation enzymatique ont montre que les conjugues flavine-oligonucleotide prepares sont bons substrats de l'enzyme et que le systeme riboflavine-nad(p)h-flavine reductase est capable d'induire la coupure d'un plasmide et celle d'un double brin lineaire d'adn.

Cinquante à 80% des patients atteints d'une lésion médullaire traumatique (LM) présente des d'épisodes d'incontinence urinaire liés à une hyperactivité détrusorienne neurogène (HDN). L’HDN est caractérisée par des contractions non inhibées du détrusor pendant la phase de remplissage vésical, qui conduit à une augmentation des pressions détrusoriennes, particulièrement lorsque l’HDN est associée à une dyssynergie vésico-sphinctérienne. L'objectif principal de la prise en charge de l’HDN est d'obtenir une vidange vésicale régulière, complète et à basse pression, ainsi que de maintenir la continence urinaire, afin d'améliorer la qualité de vie des patients et de prévenir les complications uro-néphrologiques dont l’insuffisance rénale. La prise en charge actuelle repose sur une pharmacothérapie agissant principalement au niveau de la branche efférente, motrice, du réflexe mictionnel, permettant ainsi un remplissage vésical à basse pression. Le traitement de première intention repose sur des antimuscariniques oraux, le plus souvent associés à la réalisation d’autosondages pluriquotidiens. En cas d’échec de cette thérapeutique, l’injection intradétrusorienne de toxine botulique A est proposée. Cependant, malgré leur efficacité, ces traitements induisent des effets secondaires et souffrent d’un échappement thérapeutique, conduisant à un traitement chirurgical de troisième ligne. La technique de Brindley, qui consiste en une désafférentation des racines postérieures sacrées innervant la vessie associée à une stimulation électrique, à la demande, des racines antérieures est une alternative prometteuse, mais reste peu proposée en raison de la complexité de la procédure chirurgicale requise. L'HDN résulte de l'émergence d'un réflexe spinal anormal médié par une plasticité des afférences vésicales de type-C dans les suites de la LM. Le projet de mon équipe est de réaliser une déafférentation vésicale par thérapie génique afin d'abolir le réflexe de miction spinale altérée. Dans un second temps, la miction sera déclenchée par une stimulation électrique à la demande, des neurones efférents de la vessie. Mon travail de thèse consistait à développer les outils nécessaires à une telle désafférentation moléculaire. En conséquence, j'ai construit des vecteurs défectifs HSV-1 délivrant comme transgène thérapeutique la chaine légère d’un toxine botulique (BoNT-LC), sous le contrôle du promoteur du gène codant pour la protéine liée au gène de la calcitonine (hCGRP), permettant une expression sélective au sein des neurones sensoriels. La cassette de transcription a été insérée dans le locus LAT du génome HSV-1, la seule région du génome du virus qui reste active sur le plan transcriptionnel pendant une infection latente. Ces vecteurs ont été évalués (i) in vitro, sur des lignées cellulaires d'origine neurale et sur des cultures primaires de neurones sensoriels embryonnaires et adultes de rats, ainsi que sur des cultures primaires de neurones sensoriels et sympathiques humains adultes, (ii) ex vivo, sur des cultures organotypiques de ganglions sensoriels, sympathiques et parasympathiques de rats adultes, et (iii) in vivo, post inoculation au niveau du coussinet plantaire de rats adultes. Nos résultats indiquent que (i) les vecteurs expriment des BoNT-LC fonctionnelles, clivant ainsi les protéines SNARE post infection de cultures primaires de neurones sensoriels de rats et d’être humain, et inhibant la libération du neuromédiateur CGRP dans les neurones sensoriels de rat, (ii) la sélectivité d’expression de ces vecteurs dans des neurones sensoriels humains, par rapport aux neurones sympathiques humains, et (iii) une expression transgénique prolongée in-vivo au sein de ganglions sensoriels (DRG), au moins pour trois mois, après injection. Par conséquent, ces vecteurs semblent présenter les trois principales spécifications requises pour le développement d’une future stratégie de thérapie génique visant à traiter l’HDN
Fifty to 80% of patients with traumatic spinal cord injury (SCI) undergo urinary incontinence episodes, mostly related to neurogenic detrusor overactivity (NDO). NDO is characterized by uninhibited detrusor contractions during the bladder-filling phase which could lead to a significant increase in bladder pressures, especially when associated to sphincter-destrusor-dyssynergia, leading to uro-nephrological complications. The main goal of NDO management following SCI is to achieve regular and complete bladder emptying, avoiding high intra-detrusor pressure and maintaining continence, in order to improve patients’ quality of life and to prevent renal failure. The current management is well characterized and relies on pharmacotherapy acting primarily at the level of efferent motor micturition reflex branch, thus allowing bladder filling at low pressure. First line treatment relies on oral antimuscarinics, often associated to clean intermittent bladder self-catheterization. When patients are refractory to antimuscarinics, injection of botulinum toxin A into the detrusor is proposed. However, despite their efficacy, these treatments fail to persist in the long term, leading to a third-line surgical treatment, which consists in cystoplasty augmentation or sacral neuromodulation. The Brindley technique, which consist in a sacral deafferentation of bladder posterior roots associated to an electrical stimulation, on demand, of anterior roots is a promising alternative, but remains seldom performed because of the complex surgical procedure required. NDO results from the emergence, secondary to neuronal plasticity following SCI, of an abnormal micturition reflex mediated by bladder afferent C-fibers, conveying aberrant sensory information to the spinal cord. The aim of the team where I developed my work is to silence these bladder afferent C-fibers in order to abolish the impaired spinal micturition reflex after SCI. In a second time, micturition would be fired, on demand, by electric stimulation of the bladder efferent neurons. My work consisted in developing the tools and methods required for such molecular deafferentation. Accordingly, I constructed replication-incompetent HSV-1 vectors conceived to deliver a therapeutic transcription cassette, consisting in the light chains of botulinum toxin (BoNT-LC) driven by the human version of the promoter of the gene encoding calcitonin gene-related protein (hCGRP), to achieve sensory neuron-selective transgenic expression. The transcription cassette was inserted into the LAT locus of the HSV-1 genome, the only region of the virus genome that remains transcriptionally active during latent infection. These vectors have been assessed (i) in vitro, on cell lines of neural origin and on primary cultures of rat embryonic and adult sensory neurons, and on primary cultures of adult human sensory and sympathetic neurons, (ii) ex vivo, on organotypic cultures of sensory, sympathetic and parasympathetic ganglia from adult rats, and (iii) in vivo, in sensory ganglia following infection at the hind footpad of adult rats.Our results indicate that (i) the vectors express functional BoNT-LC, thereby cleaving proteins of the SNARE complex in rat and human sensory neurons and inhibiting release of the neuromediator CGRP in rat sensory neurons, (ii) the transcription cassette delivered by the vectors display highly selectively expression towards human sensory neurons, as compared to human sympathetic neurons, and (iii) the vectors induced long-term transgenic expression in sensory (DRG) ganglia (at least for three months) following footpad injection. Therefore, the vectors seem to accomplish the three main specifications required for a future gene therapy strategy, allowing to restore urinary continence and micturition without catheterization and without any major surgery. This approach will represent a major breakthrough in the management of NDO in SCI patients with complete and incomplete lesion

Herpesviruses are large, spherical, enveloped viral particles with linear double-stranded DNA genome. Herpesvirus virion consists of an icosahedral capsid containing viral DNA, surrounded by a protein layer called tegument, and enclosed by an envelope consisting of a lipid bilayer with various glycoproteins. Herpesviruses persist lifelong in their hosts after primary infection by establishing a latent infection interrupted recurrently by reactivations. The Herpesviridae family is divided into three subfamilies; α-herpesviruses, β-herpesviruses, and γ-herpesviruses based on the genome organization, sequence homology, and biological properties. There are eight human herpes viruses: Herpes simplex virus type 1 and 2 (HSV-1, −2) andVaricella-zoster virus (VZV), which belong to the α-herpesvirus subfamily; Human cytomegalovirus (HCMV), and Human herpesvirus type 6 and 7 (HHV-6,HHV-7), which belong to the β-herpesvirus subfamily; and Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) or Human herpesvirus 8 (HHV-8), which belong to the γ-herpesvirus subfamily. Within this chapter, we summarize the current knowledge about EBV and CMV, regarding their genome organization, structural characteristics, mehanisms of latency, types of infections, mechanisms of immune escape and prevention. Epstein–Barr Virus (EBV) genome encodes over 100 proteins, of which only (30) proteins are well characterized, including the proteins expressed during latent infection and lytic cycle proteins. Based on major variation in the EBNA-2 gene sequence, two types of EBV are recognized, EBV type 1 and 2. Epstein–Barr virus types occur worldwide and differ in their geographic distribution depending on the type of virus. EBV spreads most commonly through bodily fluids, especially saliva. However, EBV can also spread through blood, blood transfusions, and organ transplantations. The EBV is associated with many malignant diseases such as lymphomas, carcinomas, and also more benign such as infectious mononucleosis, chronic active infection. The EBV has also been suggested as a trigger/cofactor for some autoimmune diseases. Overall, 1–1.5% of the cancer burden worldwide is estimated to be attributable to EBV The latently infected human cancer cells express the most powerful monogenic proteins, LMP-1 and LMP-2(Latent Membrane Protein-1,-2), as well as Epstein–Barr Nuclear Antigens (EBNA) and two small RNAs called Epstein–Barr Encoded Small RNAs (EBERs). The EBV can evade the immune system by its gene products that interfering with both innate and adaptive immunity, these include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. Currently no vaccine is available, although there are few candidates under evaluation. Human cytomegalovirus (HCMV) is a ubiquitous beta herpesvirus type 5 with seroprevalence ranges between 60 to 100% in developing countries. CMV is spread from one person to another, usually by direct and prolonged contact with bodily fluids, mainly saliva, but it can be transmitted by genital secretions, blood transfusion and organ transplantation. In addition, CMV can be transmitted vertically from mother to child. CMV infection can result in severe disease for babies, people who receive solid organ transplants or bone marrow/stem cell transplants and people with severe immune suppression such as advanced human immunodeficiency virus (HIV) infection. The HCMV has several mechanisms of immune system evasion. It interferes with the initiation of adaptive immune responses, as well as prevent CD8+ and CD4+ T cell recognition interfering with the normal cellular MHC Class I and MHC Class II processing and presentation pathways. Challenges in developing a vaccine include adeptness of CMV in evading the immune system. Though several vaccine candidates are under investigation.