Lack Of Pathogenicity Research Articles

Current Advances in Adeno-Associated Virus-Mediated Gene Therapy to Prevent Acquired Hearing Loss.

Adeno-associated viruses (AAVs) are viral vectors that offer an excellent platform for gene therapy due to their safety profile, persistent gene expression in non-dividing cells, target cell specificity, lack of pathogenicity, and low immunogenicity. Recently, gene therapy for genetic hearing loss with AAV transduction has shown promise in animal models. However, AAV transduction for gene silencing or expression to prevent or manage acquired hearing loss is limited. This review provides an overview of AAV as a leading gene delivery vector for treating genetic hearing loss in animal models. We highlight the advantages and shortcomings of AAV for investigating the mechanisms and preventing acquired hearing loss. We predict that AAV-mediated gene manipulation will be able to prevent acquired hearing loss.

Molecular Genetics and Pathogenesis of the Floating Harbor Syndrome: Case Report of Long-Term Growth Hormone Treatment and a Literature Review.

Introduction: Floating Harbor syndrome (FHS) is an extremely rare disorder, with slightly more than a hundred cases reported worldwide. FHS is caused by heterozygous mutations in the SRCAP gene; however, little is known about the pathogenesis of FHS or the effectiveness of its treatment. Methods: Whole-exome sequencing (WES) was performed for the definitive molecular diagnosis of the disease. Identified variants were validated using Sanger sequencing. In addition, systematic literature and public data on genetic variation in SRCAP and the effects of growth hormone (GH) treatment was conducted. Results: We herein report the first case of FHS in the Russian Federation. The male proband presented with most of the typical phenotypic features of FHS, including short stature, skeletal and facial features, delayed growth and bone age, high pitched voice, and intellectual impairment. The proband also had partial growth hormone deficiency. We report the history of treatment of the proband with GH, which resulted in modest improvement in growth prior to puberty. WES revealed a pathogenic c.7466C>G (p.Ser2489*) mutation in the last exon of the FHS-linked SRCAP gene. A systematic literature review and analysis of available genetic variation datasets highlighted an unusual distribution of pathogenic variants in SRCAP and confirmed the lack of pathogenicity for variants outside of exons 33 and 34. Finally, we suggested a new model of FHS pathogenesis which provides possible basis for the dominant negative nature of FHS-causing mutations and explains limited effects of GH treatment in FHS. Conclusion: Our findings expand the number of reported FHS cases and provide new insights into disease genetics and the efficiency of GH therapy for FHS patients.

Conjugal transfer of macrolide-lincosamide-streptogramin resistance from lactic acid bacteria isolated from food materials

Lactic Acid Bacteria being a reservoir of antimicrobial resistant genes, possibly pose a menace to human and animal health. Therefore, the purpose of this study was to assess the Horizontal Gene Transfer (HGT) of Macrolide-Lincosamide-Streptogramin (MLS) resistance from Lactic Acid Bacterial isolates of idli batter, chicken and sheep intestine viz., Lactobacillus fermentum (I4.16), Enterococcus hairae (C2C) and Enterococcus faecalis (SH7aab) respectively. These isolates being multidrug resistant were used as donor to analyse its transferability of MLS resistant gene to recipients Enterococcus faecalis JH2-2, Lactococcus lactis (T3.11) and Acinetobacter. Among the conjugation protocol used in the study, filter-mating method showed the efficient transfer frequency to obtain transconjugants. For all the conjugation the transconjugants obtained showed the acquisition of MLS resistance. The transfer frequency per recipient ranged between 1.4 × 10−4 to 3.7 × 10−5. The Minimum Inhibitory Concentration of erythromycin and clindamycin increased to 3–4 folds in the recipients after respective conjugation. Altogether, the results procured in this study indicates that the HGT between bacterial strains of food materials develop a vibrant gene pool that confers antibiotic resistance onto native gut microbiota even in the lack of pathogens. This probability of HGT among microbial pool should be considered as a criterion when Lactic Acid Bacteria are screened as starter cultures and probiotics in food industries.

Gene Therapy Applications of Non-Human Lentiviral Vectors.

Recent commercialization of lentiviral vector (LV)-based cell therapies and successful reports of clinical studies have demonstrated the untapped potential of LVs to treat diseases and benefit patients. LVs hold notable and inherent advantages over other gene transfer agents based on their ability to transduce non-dividing cells, permanently transform target cell genome, and allow stable, long-term transgene expression. LV systems based on non-human lentiviruses are attractive alternatives to conventional HIV-1-based LVs due to their lack of pathogenicity in humans. This article reviews non-human lentiviruses and highlights their unique characteristics regarding virology and molecular biology. The LV systems developed based on these lentiviruses, as well as their successes and shortcomings, are also discussed. As the field of gene therapy is advancing rapidly, the use of LVs uncovers further challenges and possibilities. Advances in virology and an improved understanding of lentiviral biology will aid in the creation of recombinant viral vector variants suitable for translational applications from a variety of lentiviruses.

Rapid Determination of Full and Empty Adeno-Associated Virus Capsid Ratio by Capillary Isoelectric Focusing.

Adeno-associated virus (AAV) is one of the most promising gene transfer vector types featuring long-term gene expression and low toxicity. The lack of pathogenicity and the availability of many serotypes augmented the applicability of AAV virions in gene therapy applications. The recombinant AAV capsid includes the therapeutic protein-coding transgene as well as a promoter to initiate translation and a poly A sequence portion for stabilization. Current AAV manufacturing technologies, however, cannot guarantee the generation of only full capsids, i.e., including the entire required genome. Partially filled and empty capsids are also part of the product, decreasing in this way the efficacy and safety upon clinical translation. Therefore, rapid, accurate and QC friendly analysis of the full and empty capsid ratio is of high importance during AAV vector manufacturing and release testing. In this paper, an automated capillary isoelectric focusing technique is introduced, readily applicable in the biopharmaceutical industry for fast and efficient determination of the full and empty capsid ratio. The method also reveals information about the proportion of partially filled capsids. For higher resolution (<0.1 pI unit), mixtures of wide and narrow range ampholytes were utilized. The isoelectric point and peak area percentage reproducibility (RSD) of the mixed ampholyte assay were as low as 1.67% and 2.45 %, respectively, requiring only 65 nL of sample volume per injection.

Differential Innate Immune Responses Elicited by Nipah Virus and Cedar Virus Correlate with Disparate In Vivo Pathogenesis in Hamsters.

Syrian hamsters (Mesocricetus auratus) are a pathogenesis model for the Nipah virus (NiV), and we sought to determine if they are also susceptible to the Cedar virus (CedPV). Following intranasal inoculation with CedPV, virus replication occurred in the lungs and spleens of infected hamsters, a neutralizing antibody was produced in some hamsters within 8 days post-challenge, and no conspicuous signs of disease occurred. CedPV replicated to a similar magnitude as NiV-Bangladesh in type I IFN-deficient BHK-21 Syrian hamster fibroblasts but replicated 4 logs lower in type I IFN-competent primary Syrian hamster and human pulmonary endothelial cells, a principal target of henipaviruses. The coinfection of these cells with CedPV and NiV failed to rescue CedPV titers and did not diminish NiV titers, suggesting the replication machinery is virus-specific. Type I IFN response transcripts Ifna7, Ddx58, Stat1, Stat2, Ccl5, Cxcl10, Isg20, Irf7, and Iigp1 were all significantly elevated in CedPV-infected hamster endothelial cells, whereas Ifna7 and Iigp1 expression were significantly repressed during NiV infection. These results are consistent with the hypothesis that CedPV’s inability to counter the host type I IFN response may, in part, contribute to its lack of pathogenicity. Because NiV causes a fatal disease in Syrian hamsters with similarities to human disease, this model will provide valuable information about the pathogenic mechanisms of henipaviruses.

The Adeno-associated Virus - A Safe and Promising Vehicle for Liverspecific Gene Therapy of Inherited and Non-inherited Disorders.

The first human adeno-associated virus (AAV) was originally discovered in 1960s as a contaminant of adenovirus stock preparation and thus it had not been of medical interest. Throughout the last three decades AAV has gained popularity to be used in gene therapy, mainly due to its replicative defectiveness and lack of pathogenicity in human. In addition, its ability to mediate stable and long-term expression in both non-dividing and dividing cells with specific tissue tropism makes AAV one of the most promising candidates for therapeutic gene transfer to treat many inherited as well as non-inherited disorders. Moreover, the use of AAV is not only restricted to overexpression of recombinant transgene, but also to over-express short hairpin RNA and microRNA to knockdown the expression of genes in targeted tissues. This review is organized into four parts. In the first part of the review, we discuss about the discovery and history of AAV, followed by detailed AAV biology such as virus genome, virus structure and its life cycle. In the second part of the review, the discussion is centred on the molecular mechanisms of AAV and tissue transduction, including receptor recognition and cell binding, endosomal entry, virus uncoating, nuclear entry and genome replication. Advantages and limitations of using AAV as a safe vehicle for gene delivery is also discussed. In the third part of the review, we discuss about the most commonly used AAV serotypes and variants isolated from human and non-human primates, focusing on their diverse tissue tropisms, transduction efficiency, immunological profiles and their applications in animal studies. Final part of the review focuses on the recent progress of in-vivo gene transfer using AAV for inherited and non-inherited diseases in both preclinical and clinical settings with a special emphasis on potential clinical applications of AAV in the field of liver disease.

Administration of non-pathogenic isolates of Escherichia coli and Clostridium perfringens type A to piglets in a herd affected with a high incidence of neonatal diarrhoea

A bacterial cocktail of living strains of Clostridium perfringens type A (CPA) without β2-toxin gene and non-pathogenic Escherichia coli was administered orally to newborn piglets before first colostrum intake and on 2 consecutive days on a farm with a high incidence of diarrhoea and antibiotic treatment in suckling piglets associated with E. coli and CPA. This clinical field study was driven by the hypothetic principle of competitive exclusion of pathogenic bacteria due to prior colonization of the gut mucosal surface by non-pathogenic strains of the same bacterial species with the aim of preventing disease. Although CPA strains used in this study did not produce toxins in vitro, their lack of pathogenicity cannot be conclusively confirmed. The health status of the herd was impaired by a high incidence of postpartum dysgalactia syndrome in sows (70%) and a high incidence of neonatal diarrhoea caused by enterotoxigenic E. coli and CPA during the study. No obvious adverse effect of the bacterial treatment occurred. On average, more piglets were weaned in litters treated (P=0.009). Visual pathological alterations in the small intestinal wall were more frequent in dead piglets of the control group (P=0.004) and necrotizing enteritis was only found in that group. A higher average daily weight gain of piglets in the control group (P<0.001) may be due to an increased milk uptake due to less competition in the smaller litters. The bacterial cocktail was tested under field conditions for its potential to stabilize gut health status in suckling piglets before disease development due to colibacillosis and clostridial infections; however, the gut flora stabilizing effect of the bacterial cocktail was not clearly discernible in this study. Further basic research is needed to confirm the positive effects of the bacterial treatment used and to identify additional potential bacterial candidates for competitive exclusion.

Adeno-associated virus-mediated cancer gene therapy: Current status

Gene therapy is one of the frontiers of modern medicine. Adeno-associated virus (AAV)-mediated gene therapy is becoming a promising approach to treat a variety of diseases and cancers. AAV-mediated cancer gene therapies have rapidly advanced due to their superiority to other gene-carrying vectors, such as the lack of pathogenicity, the ability to transfect both dividing and non-dividing cells, low host immune response, and long-term expression. This article reviews and provides up to date knowledge on AAV-mediated cancer gene therapy.

Gene identification and comparative molecular modeling of a Trypanosoma rangeli major surface protease

Trypanosoma rangeli is a hemoflagellate parasite which is able to infect humans. Distinct from Trypanosoma cruzi, the causative agent of Chagas disease, T. rangeli is non-pathogenic to the vertebrate host. The manner by which the T. rangeli interacts with the host is still unknown, but it certainly depends on the surface molecules. Major surface proteins (MSP) are GPI-anchored, zinc-dependent metalloproteases present in the surface of all trypanosomatids studied so far, which are implicated as virulence factors in pathogenic trypanosomatids, such as Leishmania spp and T. cruzi. The aims of this work were to generate the complete sequence of a T. rangeli MSP (TrMSP) gene and to determine the 3D-structure of the predicted protein by homology modeling. The plasmid bearing a complete copy of a TrMSP gene was completely sequenced and the predicted protein was modeled using Modeller software. Results indicate that TrMSP open reading frame (ORF) codes for a predicted 588 amino acid protein and shows all elements required for its posttranslational processing. Multiple sequence alignment of TrMSP with other trypanosomatids' MSPs showed an extensive conservation of the N-terminal and central regions and a more divergent C-terminal region. Leishmania major MSP (LmMSP), which had its crystal structure previously determined, has an overall 35% identity with TrMSP. This identity allowed the comparative molecular modeling of TrMSP, which demonstrated a high degree of structural conservation between MSPs from other trypanosomatids (TrypMSPs). All modeled MSPs have a conserved folding pattern, apart from structural divergences in the C-domain and discrete differences of charge and topology in the catalytic cleft, and present the same geometry of the canonical HEXXH zinc-binding motif. The determination of surface charges of the molecules revealed that TrMSP is a predominantly positive protein, whereas LmMSP and Trypanosoma cruzi MSP (TcMSP) are negative proteins, suggesting that substrates recognized by TcMSP and LmMSP could not interact with TrMSP. Moreover, the comparison between TrMSP and TcMSP protein sequences has revealed 45 non-neutral amino acid substitutions, which can be further assessed through protein engineering. The characteristics of TrMSP could explain, at least in part, the lack of pathogenicity of T. rangeli to humans and point to the necessity of identifying the biological targets of this enzyme.

A simplified purification method for AAV variant by polyethylene glycol aqueous two-phase partitioning

Adeno-Associated Virus (AAV) has been widely used for in vivo study and preclinical therapy due to its ability to mediate long-term transgene expression, its lack of pathogenicity and low immunogenicity. It has been found that AAV has more than ten serotypes, which each transfect certain types of cells in the viral infected organ. Current methods for purification of different AAV serotypes utilize CsCl or Iodixanol ultrahigh speed density gradient centrifugation, which is expensive and time consuming. We recently developed a simplified method, PEG/(NH4)2SO4 aqueous two phase partitioning, for purification of AAV serotype 2 and 8. The method does not require ultrahigh speed gradient centrifugation or chromatography. Here we further explore the simplified method for purification of other serotypes of AAV, serotype 6 and 9. This simplified method not only can be used to purify serotype 2 and 8, but also serotype 6 and 9, indicating that a variety of AAV serotypes can be purified by this method.

The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the in vivo gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2 cap sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids (588)QQNTA(592) of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process.

Conjugation of paclitaxel on adeno-associated virus (AAV) nanoparticles for co-delivery of genes and drugs

We have investigated the use of adeno-associated virus (AAV) nanoparticles as platforms for the co-delivery of genes and drugs to cancer cells. With its regular geometry, nanoscale dimensions, lack of pathogenicity, and high infection efficiency in a wide range of human cells and tissues, AAV is a promising vector for such applications. We tested the covalent conjugation of paclitaxel onto surface-exposed lysine residues present on the virus capsid. Immunoblotting results suggest successful attachment of drug molecules to the virus nanoparticles. Favorably, the reaction conditions did not reduce the gene delivery efficiency of the AAV vectors. Unfortunately, decrease in cancer cell viability was not observed with our AAV–taxol conjugates. For future attempts at conjugating drugs to the AAV nanoparticle, we have identified several improvements than can be considered to achieve the desired cytotoxicity in target cells.

Serotype-independent Method of Recombinant Adeno-associated Virus (AAV) Vector Production and Purification

A variety of gene transfer strategies have been developed to treat inherited, degenerative, and acquired diseases. Among the different vector systems developed so far, recombinant adeno-associated viral (AAV) vectors have shown notable benefits, including prolonged gene expression, transduction of both dividing and nondividing cells, and a lack of pathogenicity caused by wild-type infections. Thanks to these features, the use of AAV vectors as a gene transfer tool has increased dramatically during the past several years, and several recent clinical trials have used AAV vectors. However, AAV vectors are more complicated to produce than are other viral vectors. With steady advances toward clinical application, much effort has been made to isolate novel AAV serotypes and to develop methods for their efficient, scalable, and versatile production and purification. Here we review state of the art methods for AAV vector production and purification, which we have refined in our laboratory.

Suppression of adaptive immune responses during primary SIV infection of sabaeus African green monkeys delays partial containment of viremia but does not induce disease

AbstractOne of the most puzzling observations in HIV research is the lack of pathogenicity in most nonhuman primate species that are natural hosts of simian immunodeficiency virus (SIV) infection. Despite this, natural hosts experience a level of viremia similar to humans infected with HIV or macaques infected with SIV. To determine the role of adaptive immune responses in viral containment and lack of disease, we delayed the generation of cellular and humoral immune responses by administering anti-CD8– and anti-CD20 lymphocyte–depleting antibodies to sabaeus African green monkeys (Chlorocebus sabaeus) before challenge with SIVsab9315BR. In vivo lymphocyte depletion during primary infection resulted in a brief elevation of viremia but not in disease. Based on the magnitude and timing of SIV-specific CD8+ T-cell responses in the lymphocyte-depleted animals, CD8+ T-cell responses appear to contribute to viral containment in natural hosts. We found no evidence for a contribution of humoral immune responses in viral containment. These studies indicate that natural hosts have developed mechanisms in addition to classic adaptive immune responses to cope with this lentiviral infection. Thus, adaptive immune responses in natural hosts appear to be less critical for viral containment than in HIV infection.

Compensated Pathogenic Deviations: Analysis of Structural Effects

Pathogenic deviations (PDs) in humans are disease-causing missense mutations. However, in some cases, these disease-associated residues occur as the wild-type residues in functionally equivalent proteins in other species and these cases are termed ‘compensated pathogenic deviations’ (CPDs). The lack of pathogenicity in a non-human protein is presumed to be explained in most cases by the presence of compensatory mutations, most commonly within the same protein. Identification of structural features of CPDs and detection of specific compensatory events will help us to understand traversal along fitness landscape valleys in protein evolution.We divided mutations listed in the OMIM (Online Mendelian Inheritance in Man) database into PD and CPD data sets and performed two independent analyses: (i) We searched for potential compensatory mutations spatially close to the CPDs and, (ii) using our SAAPdb database, we examined likely structural effects to try to explain why mutations are pathogenic, comparing PDs and CPDs. Our data sets were obtained from a set of 245 human proteins of known structure and contained a total of 2328 mutations of which 453 (from 85 structures) were seen to be compensated in at least one functionally equivalent protein in another (non-human) species.Structural analysis results confirm previous findings that CPDs are, on average, ‘milder’ in their likely structural effects than uncompensated PDs and tend to be on the protein surface. We also showed that the residues surrounding the CPD residue in the folded protein are more often mutated than the residues surrounding an uncompensated mutation, supporting the hypothesis that compensation is largely a result of structurally local mutations.

Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus

Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.

Lack of pathogenicity and toxicity of the mycoinsecticide Metarhizium anisopliae var. acridum following acute gastric exposure in mice

Metarhizium anisopliae var. acridum, monospore culture EH-502/8 (CNRCB MaPL40), isolated in Mexico from Schistocerca piceifrons ssp. piceifrons (Orthoptera: Acrididae) was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated with one dose (108 conidia/animal) of viable (72 mice), non-viable (24 mice) conidia and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies after the inoculation. No mice showed clinical symptoms of illness or died during the study. The fungus was able to persist in some organs until day 3, but did not cause any damage to the host. The gross pathology observed was splenomegaly in mice inoculated with viable and non-viable conidia. Non-germinated conidia, observed in several organs, suggest hematogenous spread, but without any histopathological tissue reaction. Results support the non-pathogenic and non-toxic status of this fungal strain when administered in a single intragastric dose to mice.

Safety of Adeno-Associated Viral Vectors

Vectors based on recombinant adeno-associated viruses (AAVs) are being extensively explored for gene therapy owing to some of their distinguishing characteristicss such as lack of pathogenicity, wide range of infectivity and ability to provide long-term transgene expression. For many of the same reasons, recombinant AAV (rAAV) vectors have also been used as vaccine carriers to elicit immune responses against their transgene products. Extensive studies of rAAV vectors in animal models and in the clinic have revealed some safety concerns relating to their construction and production, adverse events following delivery, potential integration of the vector’s genome into host cell genomes, and the impairment of rAAV-induced CD8+ T-cell responses, which could have dire consequences for rAAV-treated individuals. Further studies to advance our knowledge of the biology of AAV and rAAV vectors are deemed necessary to allow for their more successful application in the clinic.

Cancer gene therapy using adeno-associated virus vectors

Gene therapy has offered highly possible promises for treatment of cancers, as many potential therapeutic genes involved in regulation of molecular processes may be introduced by gene transfer, which can arrest angiogenesis, tumor growth, invasion, metastasis, and/or can stimulate the immune response against tumors. Therefore, viral and non-viral gene delivery systems have been developed to establish an ideal delivery vector for cancer gene therapy over the past several years. Among the currently developed virus vectors, the adeno-associated virus (AAV) vector is considered as one of those that are closest to the ideal vector mainly for genetic diseases due to the following prominent features; the lack of pathogenicity and toxicity, ability to infect dividing and non-dividing cells of various tissue origins, a very low host immune response and long-term expression. Particularly, the most important attribute of AAV vectors is their safety profile in clinical trials ranging from CF to Parkinson's disease. Although adenovirus and several other oncolytic viruses have been more frequently used to develop cancer gene therapy, AAV also has many critical properties to be exploited for a cancer gene delivery vector. In this review, we will briefly summarize the basic biology of AAV and then mainly focus on recent progresses on AAV vector development and AAV-mediated therapeutic vectors for cancer gene therapy.