High-Capacity Adenoviral Vectors: Expanding the Scope of Gene Therapy.

The adaptation of adenoviruses as gene delivery tools has resulted in the development of high-capacity adenoviral vectors (HC-AdVs), also known, helper-dependent or "gutless". Compared with earlier generations (E1/E3-deleted vectors), HC-AdVs retain relevant features such as genetic stability, remarkable efficacy of in vivo transduction, and production at high titers. More importantly, the lack of viral coding sequences in the genomes of HC-AdVs extends the cloning capacity up to 37 Kb, and allows long-term episomal persistence of transgenes in non-dividing cells. These properties open a wide repertoire of therapeutic opportunities in the fields of gene supplementation and gene correction, which have been explored at the preclinical level over the past two decades. During this time, production methods have been optimized to obtain the yield, purity, and reliability required for clinical implementation. Better understanding of inflammatory responses and the implementation of methods to control them have increased the safety of these vectors. We will review the most significant achievements that are turning an interesting research tool into a sound vector platform, which could contribute to overcome current limitations in the gene therapy field.

Stability of the adeno-associated virus 8 reference standard material.

Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 1012 vector genome (vg)/ml, 9.38 ± 11.4 × 108 infectious units (IU)/ml and 5.76 ± 2.39 × 1011 total particles (p)/ml as determined by qPCR, TCID50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions ( <-70° C). The use of RSM is strongly recommended and endorsed by regulatory agencies to normalize laboratory internal controls and to provide accurate titration of AAV vectors lots.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations.

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

New gorilla adenovirus vaccine vectors induce potent immune responses and protection in a mouse malaria model.

BACKGROUND: A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors. RESULTS: The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen. CONCLUSION: These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor.

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 106 s-1.M-1 range, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.

Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes.

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

Post-transcriptional gene silencing of the gene encoding aldolase from soybean cyst nematode by transformed soybean roots.

Plant parasitic nematodes cause approximately 157 billion US dollars in losses worldwide annually. The soybean cyst nematode (SCN), Heterodera glycines, is responsible for an estimated one billion dollars in losses to the US farmer each year. A promising new approach for control of plant parasitic nematode control is gene silencing. We tested this approach by silencing the SCN gene HgALD, encoding fructose-1,6-diphosphate aldolase. This enzyme is important in the conversion of glucose into energy and may be especially important in actin-based motility during parasite invasion of its host. An RNAi construct targeted to silence HgALD was transformed into soybean roots of composite plants to examine its efficacy to reduce the development of females formed by SCN. The number of mature females on roots transformed with the RNAi construct designed to silence the HgALD gene was reduced by 58%. These results indicate that silencing the aldolase gene of SCN +can greatly decrease the number of female SCN reaching maturity, and it is a promising step towards broadening resistance of plants against plant-parasitic nematodes.

Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

Quality of the transgene-specific CD8+ T cell response induced by adenoviral vector immunization is critically influenced by virus dose and route of vaccination.

Adenoviral vectors have been widely used for experimental gene therapy and vaccination, yet there is a surprising lack of knowledge connecting the route and dose of adenovirus administration to the induced transgene-specific immune response. We have recently demonstrated polyfunctional CD8(+) T cells and protective memory responses using adenoviral vectors, which seem to contrast with recent reports suggesting that an exhausted CD8(+) T cell phenotype is induced by inoculation with adenoviral vectors. Accordingly, we investigated the route and dose interrelationship for transgene-specific CD8(+) T cells using adenoviral vectors encoding beta-galactosidase applied either s.c. or i.v. Irrespective of the route of inoculation, most of the adenoviral inoculum was found to disseminate systemically as the dose was raised beyond 10(9) particles. The number of transgene-specific CD8(+) T cells correlated positively with dissemination, whereas the functional capacity of the generated T cells correlated inversely with vector dissemination. A comparison of the immune response to s.c. or i.v. administration at moderate doses revealed that inoculation by both routes induced a transient peak of IFN-gamma-producing CD8(+) T cells 2 to 3 wk postinfection, but following i.v. administration, these cells were only detected in the liver. Two to four months after systemic, but not peripheral, immunization, dysfunctional transgene-specific CD8(+) T cells impaired in both cytokine production and important in vivo effector functions, accumulated in the spleen. These findings indicate that the localization of the adenoviral inoculum and not the total Ag load determines the quality of the CD8(+) T cell response induced with adenoviral vaccines.

Replication-competent lentivirus analysis of clinical grade vector products.

Lentiviral vectors are now in clinical trials for a variety of inherited and acquired disorders. A challenge for moving any viral vector into the clinic is the ability to screen the vector product for the presence of replication-competent virus. Assay development for replication-competent lentivirus (RCL) is particularly challenging because recombination of vector packaging plasmids and cellular DNA leading to RCL has not been reported with the current viral vector systems. Therefore, the genomic structure of a RCL remains theoretical. In this report, we describe a highly sensitive RCL assay suitable for screening vector product and have screened large-scale vector supernatant, cells used in vector production, and cells transduced with clinical grade vector. We discuss the limitations and challenges of the current assay, and suggest modifications that may improve the suitability of this assay for screening US Food and Drug Administration (US FDA)-licensed products.

Canine corneal fibroblast and myofibroblast transduction with AAV5.

OBJECTIVE: The aims of this study were (1) to determine the efficacy of adeno-associated vector serotype 5 (AAV5) for delivering gene therapy to canine corneal fibroblasts (CCFs) and myofibroblasts (CCMs) using enhanced green fluorescent protein (GFP) marker gene and (2) to evaluate the cytotoxicity of AAV5 to CCFs and CCMs using an in vitro model. METHODS: Healthy donor canine corneas were used to generate primary CCFs by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum-free medium containing transforming growth factor ß1 (1 ng/mL). An AAV5 titer (6.5 × 10(12) µg/mL) expressing GFP under control of hybrid cytomegalovirus + chicken ß-actin promoters (AAV5-gfp) was used to transduce CCF and CCM cultures. Delivered gene expression in CCFs and CCMs was quantified using immunocytochemistry, fluorescent microscopy, and real-time PCR. Transduction efficacy of the AAV5 vector was determined by counting DAPI-stained nuclei and EGFP-positive cells in culture. Phase-contrast microscopy, trypan blue, and dUTP nick end labeling (TUNEL) assays were used to determine the toxicity and safety of AAV5 in this canine corneal model. RESULTS: Topical AAV5 application successfully transduced a significant population of CCFs (42.8%; P < 0.01) and CCMs (28%; P < 0.01). Tested AAV5 did not affect CCF or CCM phenotype or cellular viability and did not cause significant cell death. CONCLUSIONS: The tested AAV5 is an effective and safe vector for canine corneal gene therapy in this in vitro model. In vivo studies are warranted.

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.

Requirements for baculoviruses for clinical gene therapy applications.

Baculoviruses have proven capacity for the production of recombinant proteins including virus-like particles and as viral vectors. Recent progress in preclinical studies suggest that baculoviruses have potential as new vectors for gene therapy but so far no clinical trials have been performed. To date, no specific guidelines for the use of baculoviruses as human gene therapy vectors exist but researchers can utilize existing guidelines made for other biological products. Because of the long history of research on baculoviruses, a lot of knowledge has been obtained that forms a good basis for the gene therapy development process. This article gives an overview of the current status of the application of baculovirus vectors in gene therapy and summarizes some of the challenges to overcome before the first clinical trials with baculoviruses can be accomplished.

An initiative to manufacture and characterize baculovirus reference material.

This letter to the editor brings to the attention of researchers an initiative to develop a baculovirus reference material repository. To be successful this initiative needs the support of a broad panel of researchers working with baculovirus vectors for recombinant protein production and gene delivery for either therapy or vaccination. First there is a need to reach a consensus on the nature of the reference material, the production protocols and the baculovirus characterization methods. It will also be important to define repository and distribution procedures so that the reference material is available to any researcher for calibrating experimental data and to compare experiments performed in the various laboratories. As more and more baculovirus-based products are licensed or in the final stages of development, the development of a repository of baculovirus reference material is timely. This letter describes the requirements for the reference material and for the project as a whole to be successful and calls for a partnership that would involve academic, industrial laboratories and governmental organizations to support this international initiative.

Quality control and analytical methods for baculovirus-based products.

Recombinant baculoviruses (rBac) are used for many different applications, ranging from bio-insecticides to the production of heterologous proteins, high-throughput screening of gene functions, drug delivery, in vitro assembly studies, design of antiviral drugs, bio-weapons, building blocks for electronics, biosensors and chemistry, and recently as a delivery system in gene therapy. Independent of the application, the quality, quantity and purity of rBac-based products are pre-requisites demanded by regulatory authorities for product licensing. To guarantee maximization utility, it is necessary to delineate optimized production schemes either using trial-and-error experimental setups ("brute force" approach) or rational design of experiments by aid of in silico mathematical models (Systems Biology approach). For that, one must define all of the main steps in the overall process, identify the main bioengineering issues affecting each individual step and implement, if required, accurate analytical methods for product characterization. In this review, current challenges for quality control (QC) technologies for up- and down-stream processing of rBac-based products are addressed. In addition, a collection of QC methods for monitoring/control of the production of rBac derived products are presented as well as innovative technologies for faster process optimization and more detailed product characterization.

Use of feline herpesvirus as a vaccine vector offers alternative applications for feline health.

Herpesviruses are attractive vaccine vector candidates due to their large double stranded DNA genome and latency characteristics. Within the scope of veterinary vaccines, herpesvirus-vectored vaccines have been well studied and commercially available vectored vaccines are used to help prevent diseases in different animal species. Felid alphaherpesvirus 1 (FHV-1) has been characterised as a vector candidate to protect against a range of feline pathogens. In this review we highlight the methods used to construct FHV-1 based vaccines and their outcomes, while also proposing alternative uses for FHV-1 as a viral vector.

Production of Baculovirus and Stem Cells for Baculovirus-Mediated Gene Transfer into Human Mesenchymal Stem Cells.

The discovery of the genome-editing tool CRISPR-Cas9 is revolutionizing the world of gene therapy and will extend the gene therapy product pipeline. While applying gene therapy products, the main difficulty is an efficient and effective transfer of the nucleic acids carrying the relevant information to their target destination, the nucleus of the cells. Baculoviruses have shown to be very suitable transport vehicles for this task due to, inter alia, their ability to transduce mammalian/human cells without being pathogenic. This property allows the usage of baculovirus-transduced cells as cell therapy products, thus, combining the advantages of gene and cell therapy. To make such pharmaceuticals available for patients, a successful production and purification is necessary. In this chapter, we describe the generation of a pseudotyped baculovirus vector, followed by downstream processing using depth and tangential-flow filtration. This vector is used subsequently to transduce human mesenchymal stem cells. The production of the cells and the subsequent transduction process are illustrated.

GMP-Compliant Adenoviral Vectors for Gene Therapy.

Recently, gene therapy as one of the most promising treatments can apply genes for incurable diseases treatment. In this context, vectors as gene delivery systems play a pivotal role in gene therapy procedure. Hereupon, viral vectors have been increasingly introduced as a hyper-efficient tools for gene therapy. Adenoviral vectors as one of the most common groups which are used in gene therapy have a high ability for humans. Indeed, they are not integrated into host genome. In other words, they can be adapted for direct transduction of recombinant proteins into targeted cells. Moreover, they have large packaging capacity and high levels of efficiency and expression. In accordance with translational pathways from the basic to the clinic, recombinant adenoviral vectors packaging must be managed under good manufacturing practice (GMP) principles before applying in clinical trials. Therein, in this chapter standard methods for manufacturing of GMP-compliant Adenoviral vectors for gene therapy have been introduced.

VectorMOD: Method for Bottom-Up Proteomic Characterization of rAAV Capsid Post-Translational Modifications and Vector Impurities.

Progress in recombinant AAV gene therapy product and process development has advanced our understanding of the basic biology of this critical delivery vector. The discovery of rAAV capsid post-translational modifications (PTMs) has spurred interest in the field for detailed rAAV-specific methods for vector lot characterization by mass spectrometry given the unique challenges presented by this viral macromolecular complex. Recent concerns regarding immunogenic responses to systemically administered rAAV at high doses has highlighted the need for investigators to catalog and track potentially immunogenic vector lot components including capsid PTMs and PTMs on host cell protein impurities. Here we present a simple step-by-step guide for academic rAAV laboratories and Chemistry, Manufacturing and Control (CMC) groups in industry to perform an in-house or outsourced bottom-up mass spectrometry workflow to characterize capsid PTMs and process impurities.