Abstract only
Low transduction efficiency of synovial tissues is a key concern in osteoarthritis gene therapy approaches. To determine the most efficient gene transfer strategy for effective in‐situ ...treatment of articular tissues, eight adeno‐associated viral vector (AAV) serotypes were assessed in monolayer‐cultured equine synovial cells and synovial tissue explants. Of the eight serotypes tested at 1x10
2
genome copies (GC) per cell, AAV2/2 produced the highest transduction efficiency in monolayer‐cultured cells followed by vector AAV2/5. When GC per cell was increased 10‐fold, again vector AAV2/2 produced the highest transduction efficiency followed by vector AAV2/5. However, when the same candidate vectors were used to transduce freshly‐collected synovial tissue explants, the hierarchy in transduction efficiency had changed. At approximately 3x10
2
GC per cell, vector AAV2/6.2 now produced the highest transduction efficiency followed by vector AAV2/5. When the GC per cell was increased 10‐fold, again vector AAV2/6.2 produced the highest transduction efficiency followed by vector AAV2/5. We conclude that vector AAV2/6.2 appears to be a superior vector for transduction of fibrous synovial joint tissues and that cell monolayer transduction efficiency does not accurately predict tissue transduction efficiency. Research support was provided by Brushwood Stables.
"There are now significant opportunities to improve the complex development process for gene therapies that would accelerate scientific progress and, most importantly, provide benefit to patients by ...increasing the number of effective gene therapies." The investigators plan to examine the biologic and mechanistic steps involved in AAV vector production, delivery of genes via vectors into human cells, and how therapeutic genes are activated in target cells, the results of which will provide important information to improve the efficiency of vector manufacturing and enhance the overall therapeutic benefit of AAV gene therapy. According to Vandenberghe, the small number of patients brings practical challenges in conducting trials and also limited commercial appeal-which, together, limit investments in gene therapies for these very rare indications.
Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are ...self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors.
In the recent years a plethora of AAVs from various species has become available for use as a gene delivery system. Although this research has yielded several novel promising candidates for use as a ...gene therapy tool, a significant number of these viral isolates were found inefficient for in vitro or in vivo gene transduction. This finding led us to investigate the structural nature and underlying mechanisms explaining suboptimal gene delivery.A correlation analysis of vector performance and structural data was performed on all non-human and human primate AAV previously described. We postulated that if a certain amino acid at a specific position on the AAV VP1 is entirely conserved over all AAVs except for one sequence, this particular sequence is likely to be defective due to this atypical residue, referred to as a singleton. A reversion of these singleton residues to the conserved amino-acid is then expected to yield an AAV with increased functionality.Out of the database of 150 available sequences, 30 AAV Cap clones were selected to represent the wide biodiversity as determined by extensive phylogenetic analysis on primate AAVs. Three experimental groups of clones were identified. For the first group, existing out of 14 sequences, the absence of singletons was used as the selection criterion for further evaluation. A second group of 16 sequences carries one or more singletons. A third experimental group consisted out of the sequences of the second group that were reverted to a singleton-free state by site directed mutagenesis. Transencapsidated vectors on the AAV2 packaging backbone were made for all of the members of these groups and tested for packaging capibility, in vitro transduction and in vivo gene transfer.Over 60% of the selected clones of group 1 showed functionality. More than 60% of the clones in group 2 had an improved transduction capability in vitro after the correction of one or more singletons. A number of these group 2 vectors were shown to be impaired in packaging capability. A single amino-acid substitution was able to rescue packaging to wild type levels for some clones while more singleton corrections were needed for others. A vector transencapsidated with AAVrh.8, a rhesus AAV previously shown to perform consistently well in liver, lung and muscle directed gene transfer shows a 7-fold increase in in vitro transduction. Vectors with improved transduction efficiency were selected for further evaluation in vivo by reporter gene delivery in mouse lung, muscle and liver.
ABSTRACT
Folate
receptor alpha (FRα) has been described as a factor involved in
mediating Ebola virus entry into cells
(6). Furthermore, it was
suggested that interaction with FRα results in ...internalization
and subsequent viral ingress into the cytoplasm via caveolae
(9). Descriptions of
cellular receptors for Ebola virus and its entry mechanisms are of
fundamental importance, particularly with the advent of vectors bearing
Ebola virus glycoprotein (GP) being utilized for gene transfer into
cell types such as airway epithelial cells. Thus, the ability of
FRα to mediate efficient entry of viral pseudotypes carrying GP
was investigated. We identified cell lines and primary cell types such
as macrophages that were readily infected by GP pseudotypes despite
lacking detectable surface FRα, indicating that this receptor
is not essential for Ebola virus infection. Furthermore, we find that
T-cell lines stably expressing FRα are not infectible,
suggesting that FRα is also not sufficient to mediate entry.
T-cell lines lack caveolae, the predominant route of
FRα-mediated folate metabolism. However, the coexpression of
FRα with caveolin-1, the major structural protein of caveolae,
was not able to rescue infectivity in a T-cell line. In addition, other
cell types lacking caveolae are fully infectible by GP pseudotypes.
Finally, a panel of ligands to and soluble analogues of FRα
were unable to inhibit infection on a range of cell lines, questioning
the role of FRα as an important factor for Ebola virus
entry.
A study was performed to evaluate the molecular presence of AAV in both human and nonhuman primates. PCR methodology was used to clone and characterize the variability in the cap sequence. The ...biological properties of these capsid structures were assessed using pseudotyped rAAV in gene transfer, neutralizing antibody and in vitro recepterology studies. This variety of novel genotypes led to the identifications of several capsids that showed a remarkable serology, tropism and/or gene transfer efficiency. In order to better understand this variation on a molecular level, a full phylogenetic study was performed.Phylogenetically the genus of the dependoviruses is a very heterogeneous group both structurally and biologically. Based on the capsid sequence, Adeno-Associated viruses are segregated in 6 distinct viral clades that we propose to be named Clade A through F. Members of these clades were isolated from 3 or more sources. Every clade consists of members that are biologically functional as a vector. In our sampling, some of the previously known serotypes and newly identified genotypes did not cluster into clades and are therefore denoted individually in phylogenetic three (cf. Figure).Two clades were previously uncharacterized. Clade F comprises the novel serotype AAV9. Clade C represents a group of viruses that share one single recombination event in their phylogenetic history. In 8 human subjects, 17 different viral capsids with a similar hybrid pattern were described. This finding highlights the importance of recombination as a mode of evolution for AAV.Closely related sequences to serotype AAV8 (Clade E) were retrieved from a variety of tissue in both humans and non-human primates (Baboon, Rhesus and Pigtailed macaque). This surprising finding suggests that AAV crosses species barriers either by direct transmission, by transmission through a common host or by another epidemiological mechanism that remains elusive.The importance of these findings for the gene therapy field is not to be overlooked. The combination of a wide dissemination in the human population with the ability of AAV to recombine is relevant in the evaluation of the efficacy, the immunology and the safety of AAV mediated gene transfer studies.
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