Candidate Vectors Research Articles

Gene therapy strategies for treating brain tumors: Retroviruses are still good candidates for therapeutic vectors

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. In the past few decades, many efforts have been made to improve the prognosis of GBM, however, with limited success. Many gene therapy strategies for GBM have been developed and a few have progressed to clinical trials. Retroviral vectors have superior features for gene therapy in brain cancers, including tumor specificity, immunogenicity, and longer half-life. Early gene therapy trials in GBM patients based on transplantation of retrovirus-producing cells into the brain failed to prove efficacious. Adenoviral vectors, which can be prepared as high-titer virus solutions and undergo efficient transduction in tumor cells, failed in clinical trials, likely due to immunogenicity and instability of gene expression. Alternative therapeutics such as oncolytic viruses that specifically target and destroy cancer cells are currently under investigation. In addition to novel vectors, retroviral vectors are still attractive candidates for use in gene therapy against brain tumors. Since yields of properly-packaged viral particles from virus-producing cells have been very limited so far, gene therapy by direct injection of hightiter retroviral vectors into the patients’ brains was not possible. To overcome these disadvantages, a packaging cell line that yields high-titer retroviral solutions was established by our group, enabling the direct injection of massive retroviral vector stocks directly into the brain. Mouse glioma models were effectively cured with a combination of a suicide gene/ prodrug system and a highly-concentrated retrovirus solution. Preclinical assessments, including that of replication-competent retroviruses and tumorigenicity of the combination method, have confirmed the safety of the highly-concentrated retrovirus solution. Addi tional studies are needed to address the clinical utility of such combination gene therapies. Taken together, these data suggest that retroviral vectors are still good candidates for development in gene therapy applications.

Using Non-Zero Dimensions for the Cosine and Tanimoto Similarity Search Among Real Valued Vectors

The cosine and Tanimoto similarity measures are typically applied in the area of chemical informatics, bio-informatics, information retrieval, text and web mining as well as in very large databases for searching sufficiently similar vectors. In the case of large sparse high dimensional data sets such as text or Web data sets, one typically applies inverted indices for identification of candidates for sufficiently similar vectors to a given vector. In this article, we offer new theoretical results on how the knowledge about non-zero dimensions of real valued vectors can be used to reduce the number of candidates for vectors sufficiently cosine and Tanimoto similar to a given one. We illustrate and discuss the usefulness of our findings on a sample collection of documents represented by a set of a few thousand real valued vectors with more than ten thousand dimensions.

Consensus-Building Mechanism for Setting Service Expectations in Air Traffic Flow Management

A significant challenge of effective air traffic flow management (ATFM) is to allow various competing airlines to collaborate with an air navigation service provider (ANSP) in determining flow management initiatives. Over the past 15 years this challenge has led to the development of a broad approach to ATFM known as collaborative decision making (CDM). A set of CDM principles has evolved to guide the development of specific tools that support ATFM resource allocation. However, these principles have not been extended to cover the problem of providing strategic advice to an ANSP in the initial planning stages of traffic management initiatives. This paper describes a mechanism in which competing airlines provide consensus advice to an ANSP by means of a voting mechanism. The mechanism is based on the recently developed majority judgment voting procedure. The result of the procedure is a consensus real-valued vector that must satisfy a set of constraints imposed by the weather and traffic conditions of the day in question. Although this problem was developed and modeled on the basis of specific ATFM features, it appears to be highly generic and amenable to a much broader set of applications. Analysis of this problem involved several interesting sub-problems, including a type of column generation process that created candidate vectors for input to the voting process.

Optical Flow Estimation using Phase Only-Correlation

In this work, we present an optical flow algorithm with a highly reduced set of motion vector candidates that are obtained by the Phase-Only Correlation function. For each candidate and each pixel, we estimate the cost of assigning the candidate vector to that pixel using a sophisticated filter that is robust to edges, instead of the most commonly used box filter for block matching. The proposed filter is based on the minimization of a quadratic energy function using the Gauss-Seidel optimization method. Preliminary results with images from the Middlebury database show that the proposed method reduces the angular and end-point errors by up to 25% with respect to the classic block matching method.

One-pot synthesis of PDMAEMA-bound iron oxide nanoparticles for magnetofection.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely used for multiple biomedical applications. Magnetic-assisted transfection (magnetofection) using SPION is an attractive gene vector candidate. In this work, poly(2-dimethylamino)ethyl methacrylate-bound iron oxide nanoparticles (IO-PDMAEMA) were generated using a grafting-from approach via atom transfer radical polymerization (ATRP) for use as a gene vector. Preparing an iron oxide-initiator (IO-initiator) is a typical and important step. We designed a simple method to produce an IO-initiator containing bromide groups (IO-Br). The IO-Br was synthesized by reacting iron oxide nanoparticles with 2-bromoisobutyric acid using a one-pot solvothermal method at a high temperature. We optimized IO-PDMAEMA by controlling the PDMAEMA molecular weight, allowing higher gene expression with lower cytotoxicity. The hydrodynamic diameter of IO-Br was 76.7 nm, which increased to 361.7 nm after polymerization. Transversal relaxivity studies suggested that IO-PDMAEMA can be a contrast agent for magnetic resonance imaging. The magnetofection efficacy of IO-PDMAEMA/pDNA was measured in HEK 293T cells with or without fetal bovine serum (FBS). The IO-PDMAEMA/pDNA magnetoplexes exhibited remarkably improved gene expression in the presence of a magnetic field and 10% FBS compared with a commercial product, PolyMag/pDNA. No significant cytotoxicity of IO-PDMAEMA/pDNA was observed with different incubation time periods with or without the magnetic field. Confocal laser scanning microscopic images showed that the amount of internalized plasmid DNA increased in the assisted magnetic field.

Block-matching algorithm based on harmony search optimization for motion estimation

Motion estimation is one of the major problems in developing video coding applications. Among all motion estimation approaches, Block-matching (BM) algorithms are the most popular methods due to their effectiveness and simplicity for both software and hardware implementations. A BM approach assumes that the movement of pixels within a defined region of the current frame can be modeled as a translation of pixels contained in the previous frame. In this procedure, the motion vector is obtained by minimizing a certain matching metric that is produced for the current frame over a determined search window from the previous frame. Unfortunately, the evaluation of such matching measurement is computationally expensive and represents the most consuming operation in the BM process. Therefore, BM motion estimation can be viewed as an optimization problem whose goal is to find the best-matching block within a search space. The simplest available BM method is the Full Search Algorithm (FSA) which finds the most accurate motion vector through an exhaustive computation of all the elements of the search space. Recently, several fast BM algorithms have been proposed to reduce the search positions by calculating only a fixed subset of motion vectors despite lowering its accuracy. On the other hand, the Harmony Search (HS) algorithm is a population-based optimization method that is inspired by the music improvisation process in which a musician searches for harmony and continues to polish the pitches to obtain a better harmony. In this paper, a new BM algorithm that combines HS with a fitness approximation model is proposed. The approach uses motion vectors belonging to the search window as potential solutions. A fitness function evaluates the matching quality of each motion vector candidate. In order to save computational time, the approach incorporates a fitness calculation strategy to decide which motion vectors can be only estimated or actually evaluated. Guided by the values of such fitness calculation strategy, the set of motion vectors is evolved through HS operators until the best possible motion vector is identified. The proposed method has been compared to other BM algorithms in terms of velocity and coding quality. Experimental results demonstrate that the proposed algorithm exhibits the best balance between coding efficiency and computational complexity.

Multiple candidates bit‐flipping algorithms for decoding LDPC codes

AbstractThis letter proposes an effective scheme to improve the error performance of conventional bit‐flipping (BF) algorithms for decoding low‐density parity‐check codes. The proposed scheme, which is referred to as the multiple‐candidates (MC) scheme, constructs a set of candidates for the starting vector of the iterative decoding procedure. Each candidate vector in the set is generated by randomly flipping several high error probability bits of the original hard‐decision vector. Simulation results show that two typical BF algorithms applying the MC scheme achieve noticeable gains over the original BF algorithms with moderate increase in computational cost. Copyright © 2012 John Wiley & Sons, Ltd.

Alpharetroviral Vector-mediated Gene Therapy for X-CGD: Functional Correction and Lack of Aberrant Splicing

Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91(phox)) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders.

Immunogenicity of a Recombinant Measles-HIV-1 Clade B Candidate Vaccine

Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 105 TCID50 of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.

Episomal Anchorage Maintains Non-Integrating Lentiviral Vectors in Dividing Cells

Abstract 4224Insertional mutagenesis has been a major setback in retroviral stem cell gene therapy. Vector integration events can induce alternative splicing, aberrant transcripts and read-through transcription of endogenous genes. Non-integrating vectors provide a potentially safer approach in some tissue targets. However, due to the lack of nuclear retention, the vector genome is rapidly lost during cell division and differentiation. Genomic Scaffold/Matrix Associated Regions (S/MARs) efficiently anchor the DNA to nuclear matrix proteins to generate chromosome domains in the nucleus and have been successfully used for non-viral approaches to genetic modification. Here we incorporated human ß-interferon derived S/MAR sequence between GFP reporter reading frame and 3’LTR of a non-integrating 3rd generation lentiviral transfer vector, in an effort to ‘anchor' the vector (termed ‘aLV') to the host cell genome in dividing cells. Our approach exploits two key properties of lentiviral life cycle, i.e. nuclear translocation of the viral genomic DNA and circularization to generate episomal genomes comprised of 1-LTR or 2-LTR circles. Integrating vector (iLV) and aLV (integrase-defective gag packaging plasmid) were VSV-G pseudotyped and concentrated, yielding stocks with titers of 2.3 x108 and 3×106 TU/ml, respectively. Transduction of 293T cells with aLV showed dose-dependent transduction and GFP expression similar to iLV. Transduced 293T cells were clonally expanded and maintained in vitro up to 10 weeks. A subset of 22 aLV clones and 9 iLV clones were followed over time and analyzed for transgene expression and molecular persistence of LTR circles. After >70 rounds of cell division, GFP fluorescence intensity was consistently lower, but persisted in aLV-293T clones at MFI 406.5±34.9 versus iLV at MFI 786.3±58.5. As predicted, 1-LTR and 2-LTR vector episomes were detected by PCR in genomic DNA from aLV transduced cells at the 10-week time point. Additional sequencing confirmed the presence of episomal junctions indicating the persistence of completely circularized episomes. With evidence of molecular persistence and GFP expression of S/MAR-anchored, episomal vector genomes in rapidly dividing cell lines, we next tested the performance of aLV in lineage-depleted murine hematopoietic stem and progenitor cells (mHSPCs). Overnight transduction of lin- progenitors at matched MOI yielded 96.3%±22 GFP-positive clonogenic methylcellulose colonies after iLV exposure versus 39.4%±21 after aLV exposure. Transduced mHSPCs were also transplanted into sublethally irradiated C57B/6 mice and peripheral blood leukocytes were analyzed for gene expression. Here GFP expression in the donor compartment averaged 62% for iLV compared with 10% for aLV. We are evaluating leukocyte donor subset GFP expression and will undertake secondary transplantation to formally demonstrate transduction in the stem cell compartment. In summary, lentivector mediated nuclear localization followed by beta-interferon S/MAR anchorage of the vector genome leads to episomal transgene expression in dividing target cells. Our studies suggest that S/MAR elements can serve as molecular anchors for non-integrating lentiviral episomes to provide sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo. We propose further study of aLV as a candidate vector for gene delivery to HSPCs while avoiding proviral integration and its potentially deleterious consequences. Disclosures:No relevant conflicts of interest to declare.

Novel disease-specific promoters for use in gene therapy for Parkinson's disease

Gene therapy is a promising therapeutic tool for Parkinson's disease (PD), but there is a lack of evaluated cell specific promoters that are relevant for the disease. We have chosen PD relevant promoter candidates for gene therapy vectors based on either previous studies; Drd1a, Drd2 and pDyn, or from a microarray study on parkinsonian patients; ACE, DNAJC3, GALNS, MAP1a and RNF25. These candidates have been evaluated in rat striatum to determine their suitability for use in cell specific vectors. The promoters had a neuronal specificity of 91–100%. The efficiency of the promoters was variable, but RNF25, DNAJC3 and MAP1a were comparable to widely used ubiquitous promoters. MAP1a was also affected by dopamine depletion.

Estimating Dynamic Discrete-Choice Games of Incomplete Information

We investigate the estimation of models of dynamic discrete-choice games of incomplete information, formulating the maximum-likelihood estimation exercise as a constrained optimization problem which can be solved using state-of-the-art constrained optimization solvers. Under the assumption that only one equilibrium is played in the data, our approach avoids repeatedly solving the dynamic game or finding all equilibria for each candidate vector of the structural parameters. We conduct Monte Carlo experiments to investigate the numerical performance and finite-sample properties of the constrained optimization approach for computing the maximum-likelihood estimator, the two-step pseudo maximum-likelihood estimator and the nested pseudo-likelihood estimator, implemented by both the nested pseudo-likelihood algorithm and a modified nested pseudo-likelihood algorithm.

Feeding Patterns of Potential West Nile Virus Vectors in South-West Spain

BackgroundMosquito feeding behaviour determines the degree of vector–host contact and may have a serious impact on the risk of West Nile virus (WNV) epidemics. Feeding behaviour also interacts with other biotic and abiotic factors that affect virus amplification and transmission.Methodology/Principal FindingsWe identified the origin of blood meals in five mosquito species from three different wetlands in SW Spain. All mosquito species analysed fed with different frequencies on birds, mammals and reptiles. Both ‘mosquito species’ and ‘locality’ explained a similar amount of variance in the occurrence of avian blood meals. However, ‘season of year’ was the main factor explaining the presence of human blood meals. The differences in diet resulted in a marked spatial heterogeneity in the estimated WNV transmission risk. Culex perexiguus, Cx. modestus and Cx. pipiens were the main mosquito species involved in WNV enzootic circulation since they feed mainly on birds, were abundant in a number of localities and had high vector competence. Cx. perexiguus may also be important for WNV transmission to horses, as are Cx. pipiens and Cx. theileri in transmission to humans. Estimates of the WNV transmission risk based on mosquito diet, abundance and vector competence matched the results of previous WNV monitoring programs in the area. Our sensitivity analyses suggested that mosquito diet, followed by mosquito abundance and vector competence, are all relevant factors in understanding virus amplification and transmission risk in the studied wild ecosystems. At some of the studied localities, the risk of enzootic circulation of WNV was relatively high, even if the risk of transmission to humans and horses was less.Conclusions/SignificanceOur results describe for first time the role of five WNV candidate vectors in SW Spain. Interspecific and local differences in mosquito diet composition has an important effect on the potential transmission risk of WNV to birds, horses and humans.

Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5 Å Resolution

AAV-DJ, a leading candidate vector for liver gene therapy, was created through random homologous recombination followed by directed evolution, selecting for invivo liver tropism and resistance to invitro immune neutralization. Here, the 4.5Å resolution cryo-EM structure is determined for the engineered AAV vector, revealing structural features that illuminate its phenotype. The heparan sulfate receptor-binding site is little changed from AAV-2, and heparin-binding affinity is similar. A loop that is antigenic in other serotypes has a unique conformation in AAV-DJ that would conflict with the binding of an AAV-2 neutralizing monoclonal antibody. This is consistent with increased resistance to neutralization by human polyclonal sera, raising the possibility that changed tropism may be a secondary effect of altered immune interactions. The reconstruction exemplifies analysis of fine structural changes and the potential of cryo-EM, in favorable cases, to characterize mutant or ligand-bound complexes.

Arginine functionalized peptide dendrimers as potential gene delivery vehicles

The quest for highly efficient and safe gene delivery systems has become the key factor for successful application of gene therapy. Peptide dendrimers are currently investigated as excellent candidates for non-viral gene delivery vectors. In this study, we report the synthesis and characterization of arginine functionalized peptide dendrimer-based vectors ranging from 5th generation (G5A) to 6th generation (G6A) via click chemistry, and their use for gene transfection in vitro and in vivo. The dendrimers can condense plasmid DNA (pDNA) and protect pDNAs from nuclease digestion. Both atomic force microscopy (AFM) and dynamic light scattering (DLS) revealed that the sizes of dendrimer/DNA particles were within 180–250 nm range. In vitro studies showed that the functionalized peptide dendrimers provided serum independent and high transfection efficiency on all studied cells, as over 2 fold higher than that of branched polyetherimide (PEI) in the presence of serum. Dendrimer G5A with molecular weight of 17 kDa demonstrated 6-fold transfection activity than PEI in breast tumor models, as well as good biosafety proved by in vitro and in vivo toxicity evaluation. However, G6A with molecular weight of 46 kDa showed much higher cytotoxicity. The functionalized dendrimer G5A with optimal generation may be therefore a potential candidate for gene delivery vehicle.

Buruli ulcer and current situation in Japan: A new emerging cutaneous Mycobacterium infection

Buruli ulcer (BU) is a new emerging disease and the third most common chronic mycobacterial infection in humans, caused by Mycobacterium ulcerans. Approximately 5000 cases are reported annually from at least 33 countries around the globe, but more from the tropical nations. A total of 32 cases have been reported from Japan sporadically since 1980. None of the cases were related to international travel. Of the total reported, M.ulcerans ssp. shinshuense, a subspecies speculated to be domestic to Japan or in Asia, has been isolated from 23 cases. The mode of transmission and its incubation period remain unclear, despite several proposed hypotheses, including several vectors and cutaneous wound as port of entry for the pathogen. M.ulcerans invades the skin, subcutaneous tissue, fascia and eventually forms extensive ulceration. Smear, culture, histopathology and polymerase chain reaction are established diagnostic tools to identify M.ulcerans. Multiple antimicrobial therapy is a commonly used therapeutic method, but patients often need extensive debridement and, at times, skin grafting, especially when diagnosis is delayed. Thus, expanding a system for improved awareness and diagnosis in Japan and Asia is important, together with elucidating the candidate vector and the mode of transmission. Here, to establish a base for future progress in better understanding of this infectious disease, we reviewed the characteristics of the disease together with an update of reported cases in Japan.

The AAV vector toolkit: poised at the clinical crossroads.

The discovery of naturally occurring adeno-associated virus (AAV) isolates in different animal species and the generation of engineered AAV strains using molecular genetics tools have yielded a versatile AAV vector toolkit. Promising results in preclinical animal models of human disease spurred the much awaited transition toward clinical application, and early successes in phase I/II clinical trials for a broad spectrum of genetic diseases have recently been reported. As the gene therapy community forges ahead with cautious optimism, both preclinical and clinical studies using first generation AAV vectors have highlighted potential challenges. These include cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients. A battery of second generation AAV vectors, engineered through rational and combinatorial approaches to address the aforementioned concerns, are now available. This review will provide an overview of preclinical studies with the ever-expanding AAV vector portfolio in large animal models and an update on new lead AAV vector candidates poised for clinical translation.

Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species.

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.

Evaluation of attenuated VSVs with mutated M or/and G proteins as vaccine vectors

Vesicular stomatitis virus (VSV) is a promising vector for vaccine and oncolysis, but it can also produce acute diseases in cattle, horses, and swine characterized by vesiculation and ulceration of the tongue, oral tissues, feet, and teats. In experimental animals (primates, rats, and mice), VSV has been shown to lead to neurotoxicities, such as hind limb paralysis. The virus matrix protein (M) and glycoprotein (G) are both major pathogenic determinants of wild-type VSV and have been the major targets for the production of attenuated strains. Existing strategies for attenuation included: (1) deletion or M51R substitution in the M protein (VSVΔM51 or VSVM51R, respectively); (2) truncation of the C-terminus of the G protein (GΔ28). Despite these mutations, recombinant VSV with mutated M protein is only moderately attenuated in animals, whereas there are no detailed reports to determine the pathogenicity of recombinant VSV with truncated G protein at high dose. Thus, a novel recombinant VSV (VSVΔM51-GΔ28) as well as other attenuated VSVs (VSVΔM51, VSV-GΔ28) were produced to determine their efficacy as vaccine vectors with low pathogenicity. In vitro studies indicated that truncated G protein (GΔ28) could play a more important role than deletion of M51 (ΔM51) for attenuation of recombinant VSV. VSVΔM51-GΔ28 was determined to be the most attenuated virus with low pathogenicity in mice, with VSV-GΔ28 also showing relatively reduced pathogenicity. Further, neutralizing antibodies stimulated by VSV-GΔ28 proved to be significantly higher than in mice treated with VSVΔM51-GΔ28. In conclusion, among different attenuated VSVs with mutated M and/or G proteins, recombinant VSV with only truncated G protein (VSV-GΔ28) demonstrated ideal balance between pathogenesis and stimulating a protective immune response. These properties make VSV-GΔ28 a promising vaccine vector and vaccine candidate for preventing vesicular stomatitis disease.

Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI–nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2–NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector–gene complex (PEI–nHAT–pEGFPC2–NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector–gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector–gene complexes. Considering the high transfection efficiency in the vestibular system, PEI–nHAT may be a potential vector for gene therapy of inner ear diseases, especially vestibular disorders, and deserves further study.