Viral gene delivery vectors: the next generation medicines for immune-related diseases.

Viruses have evolved to efficiently express their genes in host cells, which makes them ideally suited as gene delivery vectors for gene and immunotherapies. Replication competent (RC) viral vectors encoding foreign or self-proteins induce strong T-cell responses that can be used for the development of effective cancer treatments. Replication-defective (RD) viral vectors encoding self-proteins are non-immunogenic when introduced in a host naïve for the cognate virus. RD viral vectors can be used to develop gene replacement therapies for genetic disorders and tolerization therapies for autoimmune diseases and allergies. Degenerative/inflammatory diseases are associated with chronic inflammation and immune responses that damage the tissues involved. These diseases therefore strongly resemble autoimmune diseases. This review deals with the use of RC and RD viral vectors for unraveling the pathogenesis of immune-related diseases and their application to the development of the next generation prophylactics and therapeutics for todays' major diseases.

How Simian Virus 40 Hijacks the Intracellular Protein Trafficking Pathway to Its Own Benefit and Ours.

Viruses efficiently transfer and express their genes in host cells and evolve to evade the host's defense responses. These properties render them highly attractive for use as gene delivery vectors in vaccines, gene, and immunotherapies. Among the viruses used as gene delivery vectors, the macaque polyomavirus Simian Virus 40 (SV40) is unique in its capacity to evade intracellular antiviral defense responses upon cell entry. We here describe the unique way by which SV40 particles deliver their genomes in the nucleus of permissive cells and how they prevent presentation of viral antigens to the host's immune system. The non-immunogenicity in its natural host is not only of benefit to the virus but also to us in developing effective SV40 vector-based treatments for today's major human diseases.

Cre Recombinase Mediates the Removal of Bacterial Backbone to Efficiently Generate rSV40.

Gene therapy has been shown to be a feasible approach to treat inherited disorders in vivo. Among the currently used viral vector systems, adeno-associated virus (AAV) vectors are the most advanced and have been applied in patients successfully. An important drawback of non-integrating AAV vectors is their loss of expression upon cell division, while repeating systemic administration lacks efficacy due to the induction of neutralizing antibodies. In addition, a significant percentage of the general population is not eligible for AAV-mediated gene therapy due to pre-existing immunity. Development of additional viral vectors may overcome this hurdle. Simian virus 40 (SV40)-derived vectors have been reported to transduce different tissues, including the liver, and prevalence of neutralizing antibodies in the general population is very low. This renders recombinant SV40 (rSV40) vector an interesting candidate for effective (re-)administration. Clinical use of SV40 vectors is in part hampered by less advanced production methods compared to AAVs. To optimize the production of rSV40 and make it better suitable for clinical practice, we developed a production system that relies on Cre recombinase-mediated removal of the bacterial plasmid backbone.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

The HYSTER study: the effect of intracervically administered terlipressin versus placebo on the number of gaseous emboli and fluid intravasation during hysteroscopic surgery: study protocol for a randomized controlled clinical trial.

BACKGROUND: Transcervical resection of myoma or endometrium is a safe, hysteroscopic, minimally invasive procedure. However, intravasation of distension fluid is a common phenomenon during these procedures. In a previous study we observed venous gas emboli in almost every patient. The severity of hysteroscopic-derived embolization has been shown to be correlated to the amount of intravasation. In addition, paradoxical gas embolism, which is potentially dangerous, was observed in several patients. Studies have shown a reduction of intravasation by using intracervically administered vasopressin during hysteroscopy. We think that its analog, terlipressin, should have the same effect. In our previous research we observed more gaseous emboli as intravasation increased. Whether or not the insertion of intracervically administered terlipressin leads to a lower incidence and severity of gas embolism is unknown. We hypothesize that intracervically administered terlipressin leads to a reduction of intravasation with a lower incidence and severity of gas embolism. Terlipressin may be of benefit during hysteroscopic surgery. METHODS/DESIGN: Forty-eight patients (ASA 1 or 2) scheduled for transcervical resection of large, types 1-2 myoma or extensive endometrium resection will be included. In a double-blind fashion patients will be randomized 1:1 according to surgical treatment using either intracervically administered terlipressin or placebo. Transesophageal echocardiography will be used to observe and record embolic events. A pre- and post-procedure venous blood sample will be taken to calculate intravasation based on hemodilution. Our primary endpoint will be how terlipressin influences the severity of embolic events. Secondary endpoints include the effect of terlipressin on the amount of intravasation and on hemodynamic parameters. DISCUSSION: If terlipressin does indeed reduce the number of gaseous emboli and intravasation occurring during hysteroscopic surgery, it would be a simple method to minimize potential adverse events. It also allows for prolonged operating time before the threshold of intravasation is reached, thereby reducing the need for a second operation. TRIAL REGISTRATION: Nederlands Trial Register (Dutch Trial Register), ID: NTR5577 . Registered retrospectively on 18 December 2015.

Generation of a Vero-Based Packaging Cell Line to Produce SV40 Gene Delivery Vectors for Use in Clinical Gene Therapy Studies.

Replication-defective (RD) recombinant simian virus 40 (SV40)-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC) free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles compared to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

Autoimmune Aspects of Neurodegenerative and Psychiatric Diseases: A Template for Innovative Therapy.

Neurodegenerative and psychiatric diseases (NPDs) are today's most important group of diseases, surpassing both atherosclerotic cardiovascular disease and cancer in morbidity incidence. Although NPDs have a dramatic impact on our society because of their high incidence, mortality, and severe debilitating character, remarkably few effective interventions have become available. The current treatments, if available, comprise the lifelong intake of general immunosuppressants to delay disease progression or neurotransmitter antagonists/agonists to dampen undesired behaviors. The long-term usage of such medication, however, coincides with often severe adverse side effects. There is, therefore, an urgent need for safe and effective treatments for these diseases. Here, we discuss that many NPDs coincide with subtle chronic or flaring brain inflammation sometimes escalating with infiltrations of lymphocytes in the inflamed brain parts causing mild to severe or even lethal brain damage. Thus, NPDs show all features of autoimmune diseases. In this review, we postulate that NPDs resemble autoimmune-driven inflammatory diseases in many aspects and may belong to the same disease spectrum. Just like in autoimmune diseases, NPD symptoms basically are manifestations of a chronic self-sustaining inflammatory process with detrimental consequences for the patient. Specific inhibition of the destructive immune responses in the brain, leaving the patient's immune system intact, would be the ultimate solution to cure patients from the disease. To reach this goal, the primary targets, e.g., the primary self-antigens (pSAgs) of the patient's chronic (auto)immune response, need to be identified. For a few major NPDs, immunological studies led to the identification of the pSAgs involved in the autoimmune damage of specific brain parts. However, further research is needed to complete the list of pSAgs for all NPDs. Such immunological studies will not only provide crucial insights into NPD pathogenesis but also ultimately enable the development of a new generation of safe and effective immunotherapies for NPDs. Interventions that will dramatically improve the life expectancy and quality of life of individual patients and, moreover, will significantly reduce the health-care costs of the society in general.

Gas embolism during hysteroscopic surgery using bipolar or monopolar diathermia: a randomized controlled trial.

OBJECTIVE: The objective of the study was to determine the incidence and amount of gas embolism during hysteroscopic surgery using either monopolar or bipolar diathermia and to investigate the relationship between the severity of gas embolism and the amount of intravasation of distension fluid. STUDY DESIGN: This was a randomized, observer-blinded trial. Fifty patients, scheduled for hysteroscopic surgery, were assigned to either monopolar or bipolar diathermia. Transesophageal echocardiography was used to detect and classify gas embolism (grade 0-IV). Intravasation of distension fluid was measured. RESULTS: Venous gas embolism was observed in all but 1 patient. A higher incidence of more extensive (grade IV) was seen during bipolar diathermia (42% vs 13%; P = .031). Paradoxical embolism was observed in 2 patients. When intravasation exceeded 1000 mL, significantly more grade IV venous gas embolism was seen (P = .049). CONCLUSION: During hysteroscopic surgery, gas embolism was equally observed irrespective of the type of diathermia. However, more extensive embolism was observed when intravasation of distension fluid exceeded 1 L. These results question the acceptance of up to 2500 mL intravasation of distension fluid if bipolar diathermia is used.

Differential RNA silencing suppression activity of NS1 proteins from different influenza A virus strains.

The NS1 gene of influenza A virus encodes a multi-functional protein that plays an important role in counteracting cellular antiviral mechanisms such as the interferon (IFN), protein kinase R and retinoic acid-inducible gene product I pathways. In addition, NS1 has recently been shown to have RNA interference (RNAi) or RNA silencing suppression (RSS) activity. This study analysed the IFN antagonistic activity of NS1 and the RSS activity for several influenza subtypes: H1N1, H3N2, H5N1 and H7N7. It was shown that the various NS1 proteins were capable of inhibiting the activation of an IFN-responsive promoter. However, differential RSS activity was measured among the NS1 variants. The NS1 protein of strain A/WSN/33 (H1N1) was most potent in suppressing short hairpin RNA-mediated gene silencing. In contrast, NS1 proteins of the highly pathogenic H5N1 strains A/VN/1194/04 and A/HK/156/97 were most potent in complementing the RSS function of the human immunodeficiency virus type 1 Tat protein. These results show that the ability of NS1 to suppress RNAi varies among influenza strains and is likely to contribute to differences in viral replication capacity and pathogenicity.

HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1.

The RNA silencing pathway is an intracellular innate response to virus infections and retro-transposons. Many plant viruses counter this host restriction by RNA silencing suppressor (RSS) activity of a double-stranded RNA-binding protein, e.g., tomato bushy stunt virus P19. Here, we demonstrate P19 and HIV-1 Tat function across the plant and animal kingdoms and suppress a common step in RNA silencing that is downstream of small RNA maturation. Our experiments reveal that RNA silencing in HIV-1 infected human cells severely attenuates the translational output of the unspliced HIV-1 gag mRNA, and possibly all HIV-1 transcripts. The attenuation in gag mRNA translation is exacerbated by K51A substitution in the Tat double-stranded RNA-binding domain. Tat, plant virus RSS, or Dicer downregulation rescues robust gag translation and bolsters HIV-1 virion production. The reversal of HIV-1 translation repression by plant RSS supports the recent finding in Arabidopsis that plant miRNAs operate by translational inhibition. Our results identify common features between RNA silencing suppression of plant and animal viruses. We suggest that RNA silencing-mediated translation repression plays a strategic role in determining the viral set-point in a newly HIV-1-infected patient.

The Ebola virus VP35 protein is a suppressor of RNA silencing.

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.

Trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) can be inhibited by means of RNA silencing or interference (RNAi) using synthetic short interfering RNAs (siRNAs) or gene constructs encoding short hairpin RNAs (shRNAs) or long hairpin RNAs (lhRNAs). The use of siRNA and shRNA as antiviral therapeutic is limited because of the emergence of viral escape mutants. This problem is theoretically prevented by intracellular expression of lhRNAs generating multiple siRNAs that target the virus simultaneously, thus reducing the chance of viral escape. However, gene constructs encoding lhRNA molecules face problems with delivery to the right cells in an infected individual. In order to solve this problem, we constructed an HIV-1 variant with a 300 bp long hairpin structure in the 3' part of the genome corresponding to the Nef gene (HIV-lhNef). RESULTS: Intriguingly, HIV-lhNef potently inhibited wild-type HIV-1 production in trans. However, HIV-lhNef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous virus variants with truncated hairpin structures. Although these escape variants lost the ability to trans-inhibit HIV-1, they effectively outgrew the wild-type virus in competition experiments in SupT1 cells. CONCLUSION: Expression of the lhNef hairpin within the HIV-1 genome results in potent trans-inhibition of wild-type HIV-1. Although the mechanism of trans-inhibition is currently unknown, it remains of interest to study the molecular details because the observed effect is extremely potent. This may have implications for the development of virus strains to be used as live-attenuated virus vaccines.

Severe toxic damage to the rabbit spinal cord after intrathecal administration of preservative-free S(+)-ketamine.

BACKGROUND: Ketamine and S(+)-ketamine have been advocated for neuraxial use in the management of postoperative pain and severe intractable pain syndromes unresponsive to opioid escalation. Although clinical experience has accumulated with S(+)-ketamine, safety data on toxicity in the central nervous system after neuraxial administration of S(+)-ketamine are conflicting. In this study, neurologic and toxicologic effects on the spinal cord from repeated daily intrathecal administration of commercially available, preservative-free S(+)-ketamine were evaluated against placebo in a randomized, blinded design. METHODS: Eighteen white New Zealand rabbits were assigned to two groups receiving either 0.5 ml intrathecal S(+)-ketamine, 0.5% solution (12 rabbits), or 0.5 ml saline (6 rabbits) once a day for 7 consecutive days. During general anesthesia, an intrathecal catheter was placed between the fifth and sixth spinous processes (lumbar). Neurologic (according to Tarlov criteria) and histopathologic assessments were performed after 7 days of treatment. RESULTS: Postmortem investigation of the spinal cord and nerve roots revealed histopathologic lesions suggestive of toxic damage in 11 rabbits, from the group of 12 animals receiving S(+)-ketamine. These results were significantly different compared with 5 control animals (no histologic changes observed). There was no significant difference in neurologic status between the two groups after 7 days of intrathecal treatment. CONCLUSIONS: The authors conclude that repeated intrathecal administration of preservative-free S(+)-ketamine in a clinically relevant concentration and dosage has, considering the extent and severity of the lesions, a toxic effect on the central nervous system of rabbits.

Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1.

Recombination due to template switching during reverse transcription is a major source of genetic variability in retroviruses. In the present study we forced a recombination event in human immunodeficiency virus type 1 (HIV-1) by electroporation of T cells with DNA from a molecular HIV-1 clone that has a 300 bp long hairpin structure in the Nef gene (HIV-lhNef). HIV-lhNef does not replicate, but replication-competent escape variants emerged in four independent cultures. The major part of the hairpin was deleted in all escape viruses. In three cases, the hairpin deletion was linked to patch insertion of tRNA(asp), tRNA(glu) or tRNA(trp) sequences. The tRNAs were inserted in the viral genome in the antisense orientation, indicating that tRNA-mediated recombination occurred during minus-strand DNA synthesis. We here propose a mechanistic model for this hairpin-induced tRNA-mediated (HITME) recombination. The transient role of the cellular tRNA molecule as enhancer of retroviral recombination is illustrated by the eventual removal of inserted tRNA sequences by a subsequent recombination/deletion event.

Effect of temporary visceral ischemia on spinal cord ischemic damage in the rabbit.

BACKGROUND: Spinal cord ischemia and visceral ischemia may occur simultaneously during thoracoabdominal aortic aneurysm repair. The present rabbit study investigated the effect of a temporary interruption of the visceral perfusion on the development of ischemia-reperfusion injury of the spinal cord. METHODS: Spinal cord ischemia was induced by occlusion of the infrarenal aorta for variable durations (6 to 20 minutes) in 32 rabbits. In the visceral ischemia group, 20-minute concurrent clamping of the celiac trunk and mesenteric arteries was performed. At 24, 48, and 72 hours after ischemia, neurologic outcome was assessed in the control and visceral ischemia group. The PD50 (the duration of ischemia that produces lower limb neurologic deficits in 50% of the animals) was determined by quantal bioassay analysis. At 72 hours, histologic evaluation of spinal cord infarct size was performed. RESULTS: Compared with control animals, PD50 was significantly longer in the visceral ischemia group at 48 hours and 72 hours after ischemia. Neurologic and histologic outcomes correlated well (r = -0.90). CONCLUSIONS: The results of the present rabbit study suggest that concurrent temporary visceral ischemia does not aggravate spinal cord ischemic injury in the rabbit. Moreover, the results suggest that concurrent visceral ischemia may increase the tolerance of the spinal cord to ischemic damage.

Methodological quality of animal studies on neuroprotection in focal cerebral ischaemia.

BACKGROUND: The recurrent failure of apparently promising neuroprotective drugs to improve outcome in trials of patients with acute ischaemic stroke may partially be explained by over-optimistic conclusions about efficacy as a result of methodological shortcomings in preclinical studies. We assessed the methodological quality of animal studies of five different neuroprotective agents that have been tested in 21 clinical trials including a total of more than 12,000 patients with acute ischaemic stroke. METHODS: We performed a literature search restricted to full publications on the effects of clomethiazole, gavestinel, lubeluzole, selfotel, or tirilazad mesylate on infarct volume or functional outcome in animal models of acute focal cerebral ischaemia. We used a rating scale to assess the methodological quality of the included studies. One point was attributed to each of 10 items. A score of 4 to 6 points was considered "medium" and a score above 7 "high." RESULTS: A total of 45 articles were included. The median score on the methodological quality index was 3; 18 studies had a medium score and one a high score. Randomised treatment allocation was mentioned in 19 studies (42 %), blinded administration of study medication in 10 (22 %), and blinded outcome assessment in 18 (40 %). The study drug was administered at a median of 10 min (range, -60 to 360 min) after the onset of ischaemia. CONCLUSION: The evidence for neuroprotective efficacy that formed the basis for initiating the 21 trials was obtained in animal studies with a methodological quality that would, in retrospect, not justify such a decision. More rigorous preclinical study methodology may lead to more reliable and reproducible results.

Continuous monitoring of cerebrospinal fluid oxygen tension in relation to motor evoked potentials during spinal cord ischemia in pigs.

BACKGROUND: Perioperative assessment of spinal cord oxygenation might guide measures to prevent neurologic deficits secondary to ischemic or traumatic damage of the spinal cord. Although cerebrospinal fluid (CSF) partial pressure of oxygen (Po2) measurement has been used to detect spinal cord ischemia (SCI), the diagnostic value and the temporal resolution of CSF Po2 measurement compared with functional assessment of the spinal cord is unknown. This study compared CSF Po2 with transcranial motor evoked potentials (tcMEPs) for detection of experimental SCI. METHODS: The aorta and segmental arteries were exposed in 10 sufentanil-ketamine-anesthetized pigs (weight, 40-50 kg). Myogenic tcMEPs were recorded from the upper and lower limbs, and continuous assessment of CSF Po2 was provided by two Clark-type microcatheters inserted in the lumbar and thoracic intrathecal space. Graded lumbar SCI was produced by sequential clamping of segmental arteries. The relation between CSF Po2 and tcMEP during graded SCI was determined using linear regression. Diagnostic characteristics of CSF Po2 values for clinical SCI were determined using different cutoff points of CSF Po2. RESULTS: Lumbar CSF Po2 (baseline, 44 [interquartile range, 38-54] mmHg) decreased below 50% in all animals and was linearly related to loss of tcMEP amplitude in all animals. The median lumbar CSF Po2 during reduction of tcMEP to less than 25% of baseline was 11 (4-29) mmHg, whereas thoracic CSF Po2 remained constant (40 [28-50] mmHg). During absence of the tcMEP signal, lumbar CSF Po2 was less than 20 mmHg in 80% of the animals. Optimal sensitivity and predictive values of CSF Po2 measurement for SCI were in the range of 40-60% of baseline. CONCLUSIONS: The data indicate that intrathecal Po2 measurement is a sensitive monitoring technique to track real-time changes in local spinal cord oxygenation. Continuous monitoring of CSF Po2 might be applied for evaluation of patients who are at risk for direct or secondary SCI.

The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants.

RNA silencing comprises a set of sequence-specific RNA degradation pathways that occur in a wide range of eukaryotes, including animals, fungi and plants. A hallmark of RNA silencing is the presence of small interfering RNA molecules (siRNAs). The siRNAs are generated by cleavage of larger double-stranded RNAs (dsRNAs) and provide the sequence specificity for degradation of cognate RNA molecules. In plants, RNA silencing plays a key role in developmental processes and in control of virus replication. It has been shown that many plant viruses encode proteins, denoted RNA silencing suppressors, that interfere with this antiviral response. Although RNA silencing has been shown to occur in vertebrates, no relationship with inhibition of virus replication has been demonstrated to date. Here we show that the NS1 protein of human influenza A virus has an RNA silencing suppression activity in plants, similar to established RNA silencing suppressor proteins of plant viruses. In addition, NS1 was shown to be capable of binding siRNAs. The data presented here fit with a potential role for NS1 in counteracting innate antiviral responses in vertebrates by sequestering siRNAs.

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-2(2) from Lycopersicon esculentum.

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2(2) resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2(2) resistance gene and the susceptible allele, tm-2. The Tm-2(20 gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2(2) gene survive. The Tm-2(2) locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2(2) sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2(2) allele. Between the tm-2 and the Tm-2(2) polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2(2) gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2(2) resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2(2)-encoded resistance is aimed at the Achilles' heel of the virus.