Characterization and quality control of recombinant adenovirus vectors for gene therapy

Highly purified recombinant adenovirus undergoes routine quality controls for identity, potency and purity prior to its use as a gene therapy vector. Quantitative characterization of infectivity is measurable by the expression of the DNA binding protein, an early adenoviral protein, in an immunofluorescence bioassay on permissive cells as a potency determinant. The specific particle count, a key quality indicator, is the total number of intact particles present compared to the number of infectious units. Electron microscopic analysis using negative staining gives a qualitative biophysical analysis of the particles eluted from anion-exchange HPLC. One purity assessment is accomplished via the documented presence and relative ratios of component adenoviral proteins as well as potential contaminants by reversed-phase HPLC of the intact virus followed by protein peak identification using MALDI-TOF mass spectrometry and subsequent data mining. Verification of the viral genome is performed and expression of the transgene is evaluated in in vitro systems for identity. Production lots are also evaluated for replication-competent adenovirus prior to human use. For adenovirus carrying the human IL-2 transgene, quantitative IL-2 expression is demonstrated by ELISA and cytokine potency by cytotoxic T lymphocyte assay following infection of permissive cells. Both quantitative and qualitative analyses show good batch to batch reproducibility under routine test conditions using validated methods.     

Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro.

Cationic polymers possessing primary amine groups are inefficient in transferring nucleic acids into eukaryotic cells. With appropriate chemical modification, namely glycolylation of the amine groups of polylysine and polyallylamine, the actual number of free amino groups was decreased, hydrophilic residues were introduced, and the cytotoxicity of both polymers decreased significantly. Furthermore, in the case of polyallylamine, its ability to mediate gene transfer into cells increased by several orders of magnitude. Transfection efficiency was found to be dependent on the substitution level of amino groups and reached highest levels in the presence of lysosomotropic and/or fusogenic agents. At optimal conditions, glycolylated PAM was shown to be as efficient as the linear polyethylenimine of 22 kDa.     

Parvovirus vectors: use and optimisation in cancer gene therapy

With the increasing incidence and mortality of cancer worldwide, there is an urgent need for new therapeutic approaches. Gene therapy is one such approach and preliminary data are promising. Viral and nonviral vector systems for gene delivery are available, but most of the current systems suffer from disadvantages such as low transfection efficiencies, in vivo instability, targeting problems, mutagenic potential and immunogenicity. Viruses of the Parvoviridae family, which are characterised by their oncotropism, oncosuppression, long-term gene expression and human apathogenicity, potentially offer advantages as viral vectors. This article evaluates their usefulness in gene therapy strategies for cancer.     

Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.     

The application of DNA repair vectors to gene therapy

The nature of DNA, the sequence of the human genome and our increased understanding of the genetic basis of many inherited and acquired disorders have made the possibility of curing diseases a reality. The modulation of a host's genome is now the ultimate goal in the treatment of genetic diseases. Historically, gene therapy recognized two very different approaches: gene replacement or augmentation and gene repair. Gene repair precisely targets and corrects the chromosomal mutation responsible for a genetic and/or acquired disorder. Many recent advances have been made in this area of research.     

Bacteria as vectors for gene therapy of cancer

Anti-cancer therapy faces major challenges, particularly in terms of specificity of treatment. The ideal therapy would eradicate tumor cells selectively with minimum side effects on normal tissue. Gene or cell therapies have emerged as realistic prospects for the treatment of cancer, and involve the delivery of genetic information to a tumor to facilitate the production of therapeutic proteins. However, there is still much to be done before an efficient and safe gene medicine is achieved, primarily developing the means of targeting genes to tumors safely and efficiently. An emerging family of vectors involves bacteria of various genera. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally. Furthermore, invasive species can deliver heterologous genes intra-cellularly for tumor cell expression. Here, we review the use of bacteria as vehicles for gene therapy of cancer, detailing the mechanisms of action and successes at preclinical and clinical levels.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Summary Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro, we report on the progress of this approach and the various modifications that are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro,we report on the progress of this approach and the various modificationsthat are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Summary Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro, we report on the progress of this approach and the various modifications that are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression

A series of vectors has been developed to provide improved positive and negative selection for allelic exchange. Based on homologous regions of DNA ranging in size from less than 200 bp to over 1 kb, we have successfully used these new plasmids to introduce or remove markers in chromosomal or plasmid DNA. Wild type fimbria genes were replaced both in Salmonella enteritidis (sefA, agfA and fimC) and Escherichia coli (fasA and fasH). Regulation of 987P fimbriation could be identified after replacement of fasA and fasH with allelic reporter fusions. The expression of fasA but not fasH is dependent upon the osmolarity of the growth medium in an HNS-dependent manner, but unlike some other fimbrial systems expression is not dependent on the exogenous iron concentration.     

Avipoxviruses: infection biology and their use as vaccine vectors

Background

It has been well documented that the 5' untranslated region (5' UTR) of many positive-stranded RNA viruses contain key cis-acting regulatory sequences, as well as high-order structural elements. Little is known for such regulatory elements controlling porcine arterivirus replication. We investigated the roles of a conserved stem-loop 2 (SL2) that resides in the 5'UTR of the genome of a type II porcine reproductive and respiratory syndrome virus (PRRSV).

Results

We provided genetic evidences demonstrating that 1) the SL2 in type II PRRSV 5' UTR, N-SL2, could be structurally and functionally substituted by its counterpart in type I PRRSV, E-SL2; 2) the functionality of N-SL2 was dependent upon the G-C rich stem structure, while the ternary-loop size was irrelevant to RNA synthesis; 3) serial deletions showed that the stem integrity of N-SL2 was crucial for subgenomic mRNA synthesis; and 4) when extensive base-pairs in the stem region was deleted, an alternative N-SL2-like structure with different sequence was utilized for virus replication.

Conclusion

Taken together, we concluded that the phylogenetically conserved SL2 in the 5' UTR was crucial for PRRSV virus replication, subgenomic mRNA synthesis in particular.     

New adenovirus vectors for protein production and gene transfer

Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10–15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression. This revised version was published online in August 2006 with corrections to the Cover Date.     

Viral vectors for gene therapy: Current state and clinical perspectives

Gene therapy is the straightforward approach for the application of recent advances in molecular biology into clinical practice. One of the major obstacles in the development of gene therapy is the delivery of the effector to and into the target cell. Unfortunately, most methods commonly used in laboratory practice are poorly suited for clinical use. Viral vectors are one of the most promising methods for gene therapy delivery. Millions of years of evolution of viruses have resulted in the development of various molecular mechanisms for entry into cells, long-term survival within cells, and activation, inhibition, or modification of the host defense mechanisms at all levels. The relatively simple organization of viruses, small genome size, and evolutionary plasticity allow modifying them to create effective instruments for gene therapy approaches. This review summarizes the latest trends in the development of gene therapy, in particular, various aspects and prospects of the development of clinical products based on viral delivery systems.     

Progress and problems with the use of viral vectors for gene therapy

Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.     

Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy.

Recombinant adenoviruses with E1 sequences deleted efficiently transfer genes into a wide variety of target cells. Antigen- and nonantigen-specific responses to the therapy lead to toxicity, loss of transgene expression, and difficulties with vector readministration. We have created new cell lines that allowed the isolation of more disabled adenovirus vectors that have both E1 and E4 deletions. Studies with murine models of liver-directed gene therapy indicated that the E1- and E4-deleted vector expresses fewer virus proteins and induces less apoptosis, leading to blunted host responses and an improved safety profile. The impact of the E4 deletion on the stability of vector expression was confounded by immune responses to the transgene product, which in this study was beta-galactosidase. When transgene responses were eliminated, the doubly deleted vector was substantially more stable in mouse liver than was the E1-deleted construct. These studies indicate that adenovirus vectors with both E1 and E4 deletions may have advantages in terms of safety and efficacy over first-generation constructs for liver-directed gene therapy.     

Adeno-associated virus-based vectors in gene therapy

Adeno-associated virus (AAV) vectors were shown capable of high efficiency transduction of both dividing and nondividing cells and tissues. AAV-mediated transduction leads to stable, long-term transgene expression in the absence of apparent immune response. These properties and the broad host range of AAV vectors indicate that they constitute a powerful tool for gene therapy purposes. An additional potential benefit of AAV vectors is their ability to integrate site-specifically in the presence of Rep proteins which can be expressed transiently, thus limiting their suspected adverse effects. The major restrictions of AAV as vectors are their limited genetic capacity and strict packaging size constraint of less than 5 kb. Another difficulty is the labor-intensive and expensive procedure for the production and packaging of recombinant AAV vectors. The major benefits and drawbacks of AAV vectors and advances made in the past 3 years are discussed.     

Alphavirus expression vectors and their use as recombinant vaccines: a minireview

Alphavirus vectors have become widely used in basic research to study the structure and function of proteins and for protein production purposes. Development of a variety of vectors has made it possible to deliver foreign sequences as naked RNA or DNA, or as suicide virus particles produced using helper vector strategies. Preliminary reports also suggest that these vectors may be useful for in vivo applications where transient, high-level protein expression is desired, such as recombinant vaccines. The initial studies have already shown that alphavirus vaccines can induce strong humoral and cellular immune responses with good immunological memory and protective effects.