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At least 343 records · Page 19Linked to original sources
PubMed1997

Adenoviral preterminal protein stabilizes mini-adenoviral genomes in vitro and in vivo.

In the absence of host immunity, nonintegrating, first-generation adenoviral vectors remain stable in the nucleus of quiescent transduced cells in mice. A mini-adenoviral genome (9 kb) deleted for viral E1, E2, E3, and late genes, but containing the viral inverted terminal repeats (ITRs), transgene expression cassette (human alpha 1-antitrypsin), and the viral E4 genes was equally efficient at transducing cells in vitro or in vivo as first generation, E1-deleted vectors. In contrast to a first generation vector, gene expression as well as vector DNA was short-lived in cells transduced with the deleted adenoviral genome. We demonstrate that coexpression of the adenoviral E2-preterminal protein from the vector or in trans stabilizes the mini-genome in vitro and in vivo without evidence of cellular toxicity.

Adenoviridae↗
PubMed2006

Control of E1A under an E2F-1 promoter insulated with the myotonic dystrophy locus insulator reduces the toxicity of oncolytic adenovirus Ad-Delta24RGD.

We previously described Ad-Delta24RGD as an enhanced-infectivity oncolytic adenovirus that targets tumors with an impaired RB pathway. The common alteration of this pathway in cancer eliminates the interaction of pRB with E2F and releases free E2F to activate E2F-responsive promoters, including the E2F-1 promoter. To improve the selectivity towards RB pathway-defective tumors and reduce the toxicity of Ad-Delta24RGD we aimed to control E1A-Delta24 expression under the E2F-1 promoter. A polyA signal was inserted upstream of the E2F-1 promoter to stop transcription initiated at the adenovirus ITR and packaging signal. The human myotonic dystropy locus insulator (DM-1) was also located between the E1a enhancers and the E2F-1 promoter to further insulate the promoter. The Ad-Delta24RGD derivative containing these insulation sequences expressed less E1a-Delta24 in normal cells and resulted less toxic while maintaining the potent oncolytic activity of the parental virus. These results demonstrate that the human DM-1 inslulator can function in an adenovirus context to maintain heterologous promoter selectivity. The new oncolytic adenovirus presented here may represent a valuable therapeutic option for a broad range of tumors with a deregulated E2F/pRB pathway.

Adenoviridae↗
PubMed2002

Adeno-associated virus-mediated transfer of human acid maltase gene results in a transient reduction of glycogen accumulation in muscle of Japanese quail with acid maltase deficiency.

Glycogen storage disease type II (GSD II), Pompe's disease, is caused by the deficiency of acid alpha-D-glucosidase (GAA) in lysosome and is the most common form of GSD in Taiwan. Most cases are the infantile form. The disease is relentless and most patients die of cardiac failure and respiratory tract infection in the first year of life. At present, no treatment has been proved effective for this fatal disease. The applicability of enzyme replacement therapy is under investigation. However, high price and transient efficiency are the major problems to be solved. Accordingly, gene therapy by viral method has been conducted. In this study we constructed a plasmid that contained 5'-shortened BglII-NotI fragment human GAA cDNA, downstream of CMV promoter and bovine growth hormone polyadenylation signal, as well as AAV ITR region. When fibroblasts obtained from GSD II patients were cultured and infected with rAAV-GAA, the GAA activity of the fibroblasts increased four- to five-fold. Using acid maltase deficient (AMD) Japanese quail as the animal model, rcAAV-GAA 0.1 ml per site (1 x 10(9)-10) particles), totally 10 different sites to make 1 ml (1 x 10(1)0-11) particles), was injected into unilateral deep pectoral muscle of AMD quails. Medium (hepes) was only injected in the same way into the contralateral deep pectoral muscle to serve as control. Four days after injection, PAS staining showed disappearance of the glycogenosomes with regeneration of myocytes surrounding the intramuscular injected area as compared with the contralateral muscle of the same birds. Using anti-GAA monoclonal antibody, GAA was demonstrated on the regenerated myocytes by immunohistochemical staining and absent on the contralateral muscle of the same birds. Nevertheless, T lymphocytes infiltration was noted in both the rcAAV-GAA and hepes (medium) injected muscles and more prominent in the rcAAV-GAA-injected site. Functional evaluation demonstrated that wing flapping movement improved with wide flapping in the rAAV-GAA injected side, but not in the counterpart. Unfortunately, these histochemical and functional improvements faded away in 14 days, probably due to destruction of rcAAV by cell-mediated immunity of infiltrated T cells. Taken together, the present study suggests that rAAV can enter either human or quail cells and express and effectively reduce the glycogen accumulation in the skeletal muscle of AMD quails. These preliminary results are similar to these of low-dose rGAA replacement therapy. The mechanisms underlying the induction of cell-mediated immunity are unknown. How to elevate the number of packaged AAV, enhance the infectivity of AAV and reduce cell-mediated immunity must be solved in the future.

Animals↗
PubMed2003

Tetracycline-inducible transgene expression mediated by a single AAV vector.

Regulated gene delivery systems are usually made of two elements: an inducible promoter and a transactivator. In order to optimize gene delivery and regulation, a single viral vector ensuring adequate stoichiometry of the two elements is required. However, efficient regulation is hampered by interferences between the inducible promoter and (i) the promoter used to express the transactivator and/or (ii) promoter/enhancer elements present in the viral vector backbone. We describe a single AAV vector in which transcription of both the reverse tetracycline transactivator (rtTA) and the transgene is initiated from a bidirectional tetracycline-responsive promoter and terminated at bidirectional SV40 polyadenylation sites flanking both ITRs. Up to 50-fold induction of gene expression in human tumor cell lines and 100-fold in primary cultures of rat Schwann cells was demonstrated. In addition an 80-fold induction in vivo in the rat brain has been obtained. In vitro, the autoregulatory vector exhibits an induced expression level superior to that obtained using the constitutive CMV promoter. Although extinction of the transgene after removal of tetracycline was rapid (less than 3 days), inducibility after addition of tetracycline was slow (about 14 days). This kinetics is suitable for therapeutic gene expression in slowly progressive diseases while allowing rapid switch-off in case of undesirable effects. As compared to previously described autoregulatory tet-repressible (tetOFF) AAV vectors, the tet-inducible (tetON) vector prevents chronic antibiotic administration in the uninduced state.

Animals↗
PubMed1996

Processing of N3, a mammalian proteasome beta-type subunit.

Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5. This protein has a free N-terminus, having undergone post-translational processing. After immunoprecipitation from [35S]methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3. However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA. Therefore rapid amplification of cDNA ends ("RACE') was carried out to determine the full sequence. The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex. The half-life of the processing was determined to be 31 min in growing L-132 cells. The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies. There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits. Interestingly also, the site of cleavage of N3 (ITR decreases TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G decreases TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.

Amino Acid Sequence↗
PubMed1997

Ccmar1, a full-length mariner element from the Mediterranean fruit fly, Ceratitis capitata.

Using a PCR primer specific to the ITR sequence of a deleted mariner element we amplified a fragment of approximately 1300 bp from the genome of Ceratitis capitata. Analysis of four clones showed that they differed by approximately 4.6% in nucleotide sequence and exhibited high homology to mariner elements of the mellifera subfamily. One clone in particular, Ccmar1.18, was found to possess an ORF of 338 amino acids together with many of the features typical of mariner elements. The consensus sequence, Ccmar1, derived from these clones is presented. Maximum parsimony phylogenetic analysis of the Ccmar1 element confirms its position at the periphery of the mariner mellifera subfamily. The Ccmar1 element is estimated to be present in about 500 copies in the genome. The evolutionary history of the element in relation to the colonization history of the medfly is discussed.

Amino Acid Sequence↗
PubMed1999

The Queensland fruit fly, Bactrocera tryoni, contains multiple members of the hAT family of transposable elements.

Members of the hAT transposable element family are mobile in non-host insect species and have been used as transformation vectors in some of these species. We report that the Queensland fruit fly, Bactrocera tryoni, contains at least two types of insect hAT elements called Homer and a Homer-like element (HLE). The Homer element is 3789 bp in size and contains 12-bp imperfect inverted terminal repeats. The Homer element contains a long open reading frame (ORF) that encodes a putative transposase. Three different copies of this long ORF were recovered from the B. tryoni genome and, upon transcription and translation in an in vitro system, all produced transposase. The HLE is an incomplete element since no 3' inverted terminal repeat (ITR) was found. Homer and the HLE are as related to one another as either is to the other insect hAT elements such as Hermes, hobo, hermit and hopper. The structure and distribution of these two Homer elements is described.

Amino Acid Sequence↗
PubMed2001

The pattern electroretinogram (PERG) with contralateral corneal reference.

The pattern electroretinogram (PERG) is a sensitive test for investigation of the proximal retina. For monocular recording, the contralateral corneal reference (CCR) electrode position has been suggested as a possible alternative to the conventional ipsilateral temporal reference (ITR). We therefore compared the effect of these electrode positions on 26 subjects (median age 45.5 years, IQR 24.5-61.5). The signals were recorded monocularly with DTL nylon fibre electrodes. The visual stimuli were high contrast 19' black-white checkerboard patterns, reversing 4 times/s. PERG responses obtained from the CCR gave larger amplitudes and the increase in amplitude was statistically significant (P < 0.001). Because the CCR signal is larger, new normative ranges must be established to avoid false negative results.

Adult↗
PubMed1995

Islet cell transplantation in diabetes mellitus--from bench to bedside.

Replacement of the patient's islets of Langerhans either by pancreas transplantation or by isolated islet transplantation is the only treatment of type I diabetes mellitus to achieve an insulin-independent, constant normoglycemic state. The expense for this benefit is the need for immunosuppressive treatment of the recipient with all its potential risks. Thus, indications for pancreas or islet transplantations at present exist almost exclusively in patients with end-stage renal disease who are waiting on dialysis for a kidney graft or in diabetics with an established kidney graft obliged to be immunosuppressed. Islet transplants possess significantly potential advantages over whole-gland transplants: it is a simple procedure with only small risk, if any; it offers the potential advantages of pre-transplant reduction of immunogenicity thus possibly obviating the need for continuous life-long recipient immunosuppression and it offers the future feasibility of transplanting heterologous (pig) islets. The effectiveness of this concept was demonstrated in animal experiments and may be successfully transferred into the clinical situation. In this case, the indications for islet transplantation may be extended to non-uremic type I diabetics including diabetic children. This group of patients is the ultimate target group for this most direct and appealing concept of treating type I diabetes. As of 1994, more than 200 adult islet allografts were reported to the International Islet Transplant Registry (ITR) at the Justus-Liebig-University, Giessen, Germany. A detailed analysis of 75 well-documented cases grafted between 1990 and 1993 revealed a one-year survival rate of the patients and islets of 95% and 28% (in terms of significant basal C-peptide secretion), respectively, and insulin independence was achieved in 11% of the cases after simultaneous islet-kidney (SIK) or islet-after-kidney (IAK) transplants. At present, there is no clinical data available and the follow-up studies of the posttransplant period are too short to draw any conclusions concerning the effects of islet transplants on diabetic secondary complications. Moreover, islet transplantation is still a clinical investigational procedure. Obviously, a number of fundamental steps will have to be taken before the appealing concept of transplanting adult pancreatic islets can attain clinical importance in the treatment of type I diabetic subjects. Islet cell transplantation has come the long way from animal experiments to successful clinical application, but, more research at the bench has to be performed before islet cell transplants will be successfully performed in non-uremic, non-kidney transplanted type I diabetic patients.

Animals↗
PubMed2002

Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.

Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.

Amino Acid Sequence↗
PubMed2000

Adeno-associated virus production of soluble tumor necrosis factor receptor neutralizes tumor necrosis factor alpha and reduces arthritis.

The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.

Animals↗
PubMed2001

System for efficient helper-dependent minimal adenovirus construction and rescue.

Helper-dependent minimal adenoviral vectors deleted for all viral coding sequences are promising vectors for gene therapy. They retain only the adenovirus cis elements for replication and packaging, can accommodate up to 36 kb of foreign DNA, and exhibit prolonged transgene expression and reduced tissue toxicity as compared with first-generation adenoviral vectors. We have developed a system consisting of a set of cosmid cloning vectors (pMV and pMVX) for simple routine construction and efficient rescue of minimal adenoviral vectors. In the cloning vectors the inverted terminal repeats (ITRs) are flanked by recognition sites for the super rare-cutting endonuclease I-SceI. This allows the release of linear minimal adenovirus genomes for rescue of minimal adenovirus regardless of the sequence of the insert DNA. pMV contains a multiple cloning site for the insertion of 26 to 36 kb of therapeutic DNA. pMVX contains a noncoding human X-chromosomal DNA fragment as a vector backbone, which provides endonuclease restriction sites that allow for complete or partial replacement of the vector backbone by 1 to 26 kb of therapeutic DNA sequences, while retaining a packageable final minimal adenovirus genome size between 27 and 37.5 kb. Both vectors exist in two forms, with or without an Escherichia coli lacZ reporter gene cassette. Several minimal adenoviral vectors with insert sizes ranging from 1.5 to 16 kb were constructed with these cloning vectors. Minimal adenoviruses were efficiently rescued and amplified to high titers, using a Cre/lox-based helper system. Vectors containing the X-chromosomal backbone were stable during amplification. This simple and efficient system facilitates the construction of minimal adenoviruses and should be useful for further improvement of these new vectors.

Adenoviruses, Human↗
PubMed2002

Insect cells as a factory to produce adeno-associated virus type 2 vectors.

Recombinant adeno-associated viruses (rAAV) are produced transiently in mammalian cells usually by cotransfecting two or three plasmids containing AAV genes, adenovirus helper genes, and a vector genome. Expansion and transfection of adherent cells limit the scale of rAAV production. Efficient transfection is performed with cells on solid support media such as tissue culture plates. A large animal study or a human clinical trial may require 10(15) particles of vector, depending on dose. To generate this quantity of rAAV by transfection, more than 10(11) HEK293 cells may be needed, which would require about 5000 x 175 cm(2) flasks. The ability to scale up rAAV production by these methods severely restricts the commercialization and use of AAV vectors. A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus is widely employed for large-scale production of heterologous proteins in cultured insect cells and may provide an attractive alternative. Toward this goal, we have explored the production of rAAV in invertebrate cells. Sf9 cells may be coinfected in suspension cultures with three recombinant baculoviruses (a Rep-baculovirus, a VP-baculovirus, and an AAV ITR vector genome baculovirus) and, 3 days later, rAAV is recovered. The particles produced are indistinguishable from 293 cell-produced rAAV, as determined on the basis of physical properties and biologic activities. Particles produced by either method were composed of similar proteins and nucleic acid. The yield of genome-containing particles produced per Sf9 cell approached 5 x 10(4), thus, 1000 ml of cultured Sf9 cells produced the equivalent of between 500 to 1000 x 175 cm(2) flasks of 293 cells. This robust system provides a simple, cost-effective method for AAV vector production.

Animals↗
PubMed2003

Tight positive regulation of transgene expression by a single adenovirus vector containing the rtTA and tTS expression cassettes in separate genome regions.

We previously developed single adenovirus (Ad) vectors that contained the components for a tetracycline-regulatable gene-expression system in the E1 and E3 deletion regions, and showed that the Ad vectors containing the tet-on system exhibit a much inferior regulation of transgene expression than those containing the tet-off system. In many cases, the tet-on system may be preferable because of its positive regulation of transgene expression. To this end, in the present study, by introducing the latest generation reverse tetracycline-responsive transcriptional activator (rtTA2s-M2 or rtTA2s-S2) and the tetracycline-controlled transcriptional silencer (tTS) into the original tet-on system, we constructed various modified Ad-mediated tet-on systems. Among them, the novel single Ad vector, which contained three heterologous gene-expression cassettes of the gene of interest, rtTA2s-S2, and tTS in the E1 deletion region, the E3 deletion region, and the region between E4 and 3'ITR, respectively, displayed vastly improved doxycycline-inducible gene expression in terms of low basal expression, high induced expression, and high responsiveness to doxycycline both in vitro and in vivo. These results also suggest that the low responsiveness to doxycycline may explain why the original tet-on system in the context of the Ad vector is not effective in vivo. This is the first report describing the cloning of three heterologous gene-expression cassettes into three separate regions of the E1/E3-deleted Ad genome. This improved Ad-mediated tet-on system might be useful for gene therapy and greatly facilitate the analyses of gene function.

Adenoviridae↗
PubMed1996

Quantitative analysis of the packaging capacity of recombinant adeno-associated virus.

Recombinant adeno-associated viruses (AAV) are among the most promising vectors for gene therapy of genetic diseases, including cystic fibrosis (CF). However, because of its small genome size, the capacity of AAV to package a therapeutic gene is limited. The efficiency of packaging the cystic fibrosis transmembrane conductance Regulator (CFTR) gene into AAV will be an important factor in determining whether recombinant AAV can be developed as a vector for transferring CFTR cDNA to the airway epithelia of patients with CF. Current understanding of the AAV biology suggests that AAV can package a genome slightly larger than the size of a wild-type genome. The precise range of the genome size and the efficiency of packaging have not been defined. Using a series of AAV vectors with progressively-increasing genome size, we were able to analyze quantitatively the packaging efficiency in relation to the vector size and to determine the size limit for packaging. The packaging efficiencies of AAV vectors of variable sizes were determined directly by assaying DNA contents of viral particles, and indirectly by analyzing their efficiency in transfer of a chloramphenicol acetyltransferase (CAT) reporter gene into target cells. Our studies showed that the optimal size of AAV vector is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, up to 5.2 kb, the packaging efficiencies in this large size range were sharply reduced. When the AAV genome size was smaller than 4.1 kb, the packaging efficiency was also suboptimal. In contrast, when the size of the genome was less than half the length of the wild-type genome, two copies of the vector were packaged into each virion, suggesting that the copy number control during packaging is a "head-full" mechanism. Because the length of the minimal cDNA of CFTR is about 4.5 kb, these results suggest it is possible to package the CFTR gene into AAV if the combined length of transcriptional elements and ITRs is kept under 500 bp. The results of this study are important for directing the design of AAV vectors for efficient gene transfer, as well as for a better understanding of the mechanism of AAV genome packaging.

Blotting, Southern↗
PubMed1997

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.

Animals↗
PubMed1997

Modulation of the specificity and activity of a cellular promoter in an adenoviral vector.

Most gene therapy studies with recombinant adenoviruses employ viral promoters and lack tissue specificity. To determine whether a tissue-specific cellular promoter inserted into the adenoviral genome can direct the expression of a reporter gene in a tissue-specific manner, recombinant adenoviruses containing a nuclear lacZ gene driven by a human ventricular/slow muscle myosin light chain 1 promoter with and without a muscle creatine kinase enhancer were constructed. The ability of these viruses to express the reporter genes in infected myogenic and nonmyogenic cell lines was studied. Intramuscular injection of these viruses into mice showed that little or no reporter gene expression occurred in muscle fibers, although a relatively high level of lacZ gene expression was observed in surrounding connective tissue. Insertion of adenovirus sequences from the 5' inverted terminal repeat (ITR) region and/or the protein IX region into plasmids resulted in decreased reporter gene expression from myosin light chain 1 promoter in transfected C2C12 myotubes and 293 cells, as well as in injected muscles. These results suggested that negative elements are present in the adenoviral genome. This negative effect seems neither tissue nor species specific. Adenovirus cis-elements that may affect the specificity and activity of a cellular promoter are discussed.

Adenoviridae↗
PubMed1998

Factors influencing recombinant adeno-associated virus production.

Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary, this study describes a general method to titrate rAAV, independently of the transgene and its expression, and to measure the level of contamination with adenovirus and rep-positive AAV. Furthermore, we report a new production procedure using adenoviral plasmids instead of virions and resulting in rAAV stocks with undetectable adenovirus contamination.

Animals↗