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At least 1,135 records · Page 63Linked to original sources
PubMed2002

Use of a chimeric adenovirus vector enhances BMP2 production and bone formation.

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Adenoviridae↗
PubMed2002

A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors.

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

B-Lymphocytes↗
PubMed2004

Infectious titer assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events.

A highly sensitive assay for determination of infectious titers of recombinant adeno-associated virus (AAV) by limiting dilution analysis is described. This assay is capable of detecting single infectious events and can therefore provide an absolute rather than relative measure of infectivity. The assay utilizes a HeLa-derived AAV2 Rep/Cap-expressing cell line, D7-4, grown in 96-well plates and infected with replicate 10-fold serial dilutions of AAV2 vectors in the presence of adenovirus type 5. Forty-eight hours after infection, vector genome replication is determined by quantitative PCR (Q-PCR). A linear relationship between vector genome input and replicated copy number (slope = 2670 copies per vector genome) was determined, enabling detection of one infectious event per well by Q-PCR. The observed binomial distribution of the end-point data confirmed that single infectious events could be detected, and allowed calculation of infectious titers by the Kärber method. Analysis of an AAV2 reference vector, AAV-hFIX16, in 21 independent determinations gave an average ratio of AAV vector genomes (VG) to infectious units (IU) of 8.3 +/- 4.2 VG/IU, a value close to the theoretical limit. No significant differences in vector particle-to-infectious unit ratios were observed between vectors purified by column chromatography (9.3 +/- 5.0 VG/IU, n = 7) and cesium chloride gradient ultracentrifugation (6.4 +/- 3.2 VG/IU, n = 7).

Cell Line, Tumor↗
PubMed2004

Comparison of adenoviral and adeno-associated viral vectors for pancreatic gene delivery in vivo.

Although effective gene therapy vectors have been developed for organ systems such as the liver, an effective delivery vector to the pancreas in vivo has remained elusive. Of the currently available viral vectors, adenovirus and adeno-associated virus (AAV) are two of the most efficient at transducing nondividing cells. We have constructed recombinant adenovirus (AdVLacZ), adeno-associated virus serotype 2 (AAV2LacZ), and pseudotyped adeno-associated virus serotype 5 and 8 (AAV5LacZ, AAV8LacZ) carrying the LacZ reporter, and compared the transduction efficiency of these four vectors in the pancreas of mice in vivo. We showed that adenovirus, AAV2, and AAV8 are capable of transducing the pancreas in vivo, but with different expression kinetics, efficiencies of transduction, and persistence. AdVLacZ-transduced pancreas exhibited maximum LacZ expression at 1 week postdelivery, with greater than 90% of expression lost at 4 weeks. AAV2LacZ-transduced pancreas displayed peak LacZ levels at 4 weeks postdelivery, with no significant decrease in expression for up to 8 weeks. AAV8LacZ was at least 10-fold more efficient than AAV2LacZ in transducing the pancreas in vivo, with significant levels of expression detectable at 1 week, whereas AAV5LacZ did not result in any detectable transgene expression at all tested time points. All three vectors primarily transduced pancreatic acinar cell types, with limited transduction of pancreatic endocrine cells. AdVLacZ elicited a significant leukocyte infiltration early after delivery into the pancreas, whereas none of the AAV vectors elicited a significant leukocyte response. None of the tested vectors caused significant changes in serum amylase or blood glucose levels, suggesting that they do not significantly alter pancreatic function. These vectors will be useful for studying novel gene delivery based treatments in animal models for diabetes and other pancreatic disorders.

Adenoviridae↗
PubMed1999

Gene transfer into the liver of nonhuman primates with E1-deleted recombinant adenoviral vectors: safety of readministration.

Preclinical studies were designed to investigate the safety of recombinant adenoviruses infused into the portal vein of adult rhesus monkeys, as well as the safety and efficacy of readministration of these agents. The vectors used were recombinant adenoviruses, the E1 region of which was replaced with a marker gene expression cassette. Four 3- to 5-kg rhesus monkeys underwent portal vein cannulation, and infusion of escalating doses of recombinant first-generation vector. Serial sequential liver biopsies were performed, and necropsies were performed out to 14 months. X-Gal histochemical analysis of the liver showed evidence of dose-dependent increased gene transfer throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was also present in transduced livers, and occurred in a dose-dependent manner. Severe toxicity, including mortality, was noted at the highest dose of vector. Readministration of a second vector was associated with the same degree of toxicity as the first vector, but prompted a much more vigorous neutralizing antibody response. The data suggest that intraportal administration and readministration of recombinant adenoviral E1-deleted vectors are feasible and safe. Vector administration at the highest dose (1 x 10(13) particles/kg) was associated with severe clinical and biochemical toxicity, and significant gene expression was associated with transaminitis. Readministration of vector is safe, but gene transfer is limited by the presence of neutralizing antibody.

Adenoviridae↗
PubMed1999

Increased duration of transgene expression in the lung with plasmid DNA vectors harboring adenovirus E4 open reading frame 3.

For gene therapy to be effective in the treatment of chronic diseases, plasmid DNA (pDNA) vectors that provide persistent expression of therapeutic levels of the transgene product are desirable. Studies in the lung with adenovirus vectors showed that products of the adenovirus E4 region can act both in cis and in trans to increase the duration of expression when transcription of the transgene was under the control of the human cytomegalovirus (CMV) promoter. To determine if these E4-encoded proteins could also effect greater persistence of expression from a nonviral vector, a complex composed of cationic lipid GL-67, a CMV promoter plasmid (pCF1-CAT), and an E4-containing adenovirus vector (Ad2/betagal-4) was instilled into the lungs of BALB/c nu/nu mice. Significant increases in the duration of transgene expression were observed for up to 10 weeks postinstillation compared with expression from mice instilled with control complexes containing an adenovirus vector deleted of most of E4 (Ad2/betagal-2). This effect could also be observed in immunodeficient NIH-rnu rats as well as in immunocompetent BALB/c mice. Studies with CMV promoter mutants indicated that a region proximal to the promoter was necessary for the E4-mediated increase in longevity of expression. In addition to the CMV promoter, a CMV enhancer-human mucin I (MUC-I) hybrid promoter also responded to these E4-encoded proteins with increased persistence of transgene expression, but a human interleukin 8 (IL-8) promoter did not. Ad2/betagal-4 could be replaced by a pDNA vector expressing only the E4 region, indicating that products of the E4 region alone were sufficient in the absence of expression from the rest of the adenovirus genome. Further analysis indicated that the protein encoded by open reading frame 3 (ORF3) alone was sufficient for conferring the increase in persistence of expression. These data indicate that expression of a single protein from the adenovirus genome can significantly improve the duration of transgene expression from pDNA vectors, and increases the feasibility of using nonviral vectors for the treatment of chronic diseases.

Adenoviridae↗
PubMed1999

Influence of sequence and size of DNA on packaging efficiency of parvovirus MVM-based vectors.

We have derived a vector from the autonomous parvovirus MVM(p), which expresses human IL-2 specifically in transformed cells (Russell et al., J. Virol 1992;66:2821-2828). Testing the therapeutic potential of these vectors in vivo requires high-titer stocks. Stocks with a titer of 10(9) can be obtained after concentration and purification (Avalosse et al., J. Virol. Methods 1996;62:179-183), but this method requires large culture volumes and cannot easily be scaled up. We wanted to increase the production of recombinant virus at the initial transfection step. Poor vector titers could be due to inadequate genome amplification or to inefficient packaging. Here we show that intracellular amplification of MVM vector genomes is not the limiting factor for vector production. Several vector genomes of different size and/or structure were amplified to an equal extent. Their amplification was also equivalent to that of a cotransfected wild-type genome. We did not observe any interference between vector and wild-type genomes at the level of DNA amplification. Despite equivalent genome amplification, vector titers varied greatly between the different genomes, presumably owing to differences in packaging efficiency. Genomes with a size close to 100% that of wild type were packaged most efficiently with loss of efficiency at lower and higher sizes. However, certain genomes of identical size showed different packaging efficiencies, illustrating the importance of the DNA sequence, and probably its structure.

Bacteriophage lambda↗
PubMed1999

Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines.

We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.

Adenoviridae↗
PubMed2002

Lentiviral vectors for gene delivery into cells.

Human immunodeficiency virus type I (HIV) is the etiologic agent of acquired immunodeficiency syndrome or AIDS. Vectors based upon HIV have been in use for over a decade. Beginning in 1996, with the demonstration of improved pseudotyping using vesicular stomatitis virus (VSV) G protein along with transduction of resting mammalian cells, a series of improvements have been made in these vectors, making them both safer and more efficacious. Taking a cue from vector development of murine leukemia virus (MLV), split coding and self-inactivating HIV vectors now appear quite suitable for phase I clinical trials. In parallel, a number of pre-clinical efficacy studies in animals have demonstrated the utility of these vectors for various diseases processes, especially neurodegenerative and hematopoietic illnesses. These vectors are also appropriate for the study of other viruses (specifically of viral entry) and investigation of the HIV replicative cycle, along with straightforward transgene delivery to target cells of interest. Vectors based upon other lentiviruses have shown similar abilities and promise. Although concerns remain, particularly with regards to detection and propagation of replication-competent lentivirus, it is almost certain that these vectors will be introduced into the clinic within the next 3-5 years.

Animals↗
PubMed1990

Transcription vectors that facilitate the identification and mapping of RNA splice sites in genomic DNA.

Two transcription vectors were constructed that can identify the splice sites at exon-intron boundaries of inserted DNA fragments possessing the complementary splice site. One vector contains the 5' splice donor site and flanking exon-intron sequences from the 3' end of the adenovirus first late leader. The other vector contains the 3' splice acceptor site and the branch acceptor site, plus the flanking exon-intron sequences from the 5' end of the adenovirus second late leader. Both vectors contain a multiple cloning site for insertion of DNA fragments. DNA fragments supplying the complementary splice site, including the adjacent exon and intron sequences, were inserted into the vectors. The vectors were used as templates for the synthesis of chimeric RNA transcripts that were spliced in in vitro splicing extracts. Chimeric transcripts from the vectors containing complementary splice site boundary regions from the human growth hormone gene were accurately spliced in vitro. A splice site from a human growth hormone intron that is not normally spliced in vitro was spliced when paired with an adenovirus splice site. These vectors can be used to identify splice sites and to determine the lengths of exons and their attached introns within a DNA fragment of unknown coding content.

Base Sequence↗
PubMed1993

High-efficiency gene transfer to primary monkey airway epithelial cells with retrovirus vectors using the gibbon ape leukemia virus receptor.

The efficiency of retrovirus-mediated gene transfer to primary airway epithelial cells from rhesus monkeys was evaluated. We compared the use of murine amphotropic retrovirus vectors to the use of murine retrovirus vectors containing the envelope (Env) glycoproteins from gibbon ape leukemia virus (GALV). These vectors use distinct receptors to gain entry into host cells. We found that vectors with the GALV Env glycoproteins are up to 10-fold more efficient at transducing genes into primary monkey airway epithelial cells than vectors with the amphotropic Env glycoproteins. Under optimal conditions, up to about 80% of primary monkey airway epithelial cells could be transduced with the vector containing the GALV Env glycoproteins. In addition, we found that delivery of retrovirus vectors to the apical side of polarized airway epithelial cultures was significantly more efficient than delivery to the basal side. These results suggest the feasibility of luminal delivery of retrovirus vectors to the lung.

Animals↗
PubMed1995

Protection of retroviral vector particles in human blood through complement inhibition.

The rapid inactivation of murine-derived retroviral vectors in human or nonhuman primate sera is largely attributed to the activity of complement mediated through the classical pathway. In this study, we have further investigated the relationship between the human complement cascade and retrovirus inactivation. Preincubation in normal human serum effectively inactivated LXSN retroviral vector particles, whereas the vector maintained the ability to transduce cells following incubation in sera deficient in either the C1, C2, C3, C5, C6, C8, or C9 human complement proteins. Preincubation of serum with monoclonal antibodies (mAbs) that functionally block specific complement components, including C5, C6, C8, and C9, successfully protected the LXSN vector from complement-mediated inactivation. Treatment of serum with cobra venom factor, which consumes terminal complement, also effectively protected the vector from inactivation. LXSN vector survival in serum corresponded inversely to the level of complement activity following treatment of serum with anti-C5 mAb as assessed in an erythrocyte hemolytic assay. Additionally, pretreatment of human whole blood with anti-C5 mAb effectively inhibited inactivation of the LXSN vector. Taken together, these data demonstrate that formation of the membrane attack complex (MAC, C5b-9) is required for the inactivation of the murine-based LXSN retroviral vector in human blood and that this process can be abrogated with the use of soluble complement inhibitors.

3T3 Cells↗
PubMed1995

The effects of human serum and cerebrospinal fluid on retroviral vectors and packaging cell lines.

Human serum is known to inactivate many retroviruses, including murine leukemia viruses (MLV). Exposure of vectors based on MLV to human serum components would presumably decrease the efficiency of gene transfer in vivo. Human serum also lyses xenogeneic cells, which would affect the survival of retroviral vector packaging cells in vivo. The effects of other body fluids, such as cerebrospinal fluid (CSF), on MLV vectors and packaging lines have not been studied. We have found that retroviral vectors packaged in ecotropic, amphotropic, and gibbon ape leukemia virus (GALV) envelope proteins were all inactivated by human sera, and human sera also lysed mouse NIH-3T3 cells and the retroviral vector packaging cells derived from them. Human fibroblasts producing amphotropic vector particles were resistant to lysis, but the particles produced by them were inactivated. In contrast, CSF did not inactivate MLV vectors, nor did it lyse murine retrovirus packaging cells. Our results suggest that exposure to human serum may prevent in vivo gene transfer by MLV vectors and xenogeneic packaging lines, but gene transfer within the central nervous system should be more successful.

3T3 Cells↗
PubMed1995

R-region cDNA inserts in retroviral vectors are compatible with virus replication and high-level protein synthesis from the insert.

Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by the retroviral promoter. However, the typical retroviral vector design places the cDNA downstream of the retroviral packaging signal and far from the retroviral promoter. In an attempt to improve protein production levels from cDNAs expressed in retroviral vectors, we inserted the MyoD or the purine nucleoside phosphorylase (PNP) cDNAs into the R regions of both retroviral LTRs, close to the retroviral promoter and just upstream of the polyadenylation signal present in each long terminal repeat (LTR). These R-region double-copy vectors could be produced in unrearranged form, although the titer was about seven-fold lower than that of typical vectors. R-region positioning of the MyoD cDNA resulted in five-fold higher MyoD expression compared to MyoD expression in a typical vector, whereas PNP expression was not improved. Thus, R-region double-copy vectors provide an alternative vector design that can improve protein expression from some cDNAs.

Animals↗
PubMed1996

"Sero-switch" adenovirus-mediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype.

Recombinant, replication-deficient adenovirus (Ad) vectors have been successfully used to transfer and express the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA in vivo in the respiratory epithelium of experimental animals and humans with cystic fibrosis (CF). Since Ad-directed gene expression wanes over time, repeat administration is necessary to achieve an effective treatment for CF. A major hurdle to such a strategy is the possibility that anti-Ad humoral immunity may prevent gene expression in individuals with pre-existing anti-Ad immunity or following repeat administration. One strategy to circumvent such a problem would be alternating the use of Ad vectors belonging to different subgroups. Neutralizing antibodies developed with the administration of one Ad serotype do not cross-react with an Ad belonging to a second serotype in a manner that blocks infection and gene expression. To test this hypothesis, an immunizing dose of wild-type Ad5 (subgroup C), Ad4 (subgroup E), or Ad30 (subgroup D) was administered intratracheally to experimental animals, followed by an intratracheal administration of a replication-deficient subgroup C-derived vector coding for marker genes (chloramphenicol acetyl transferase or beta-galactosidase) or for the normal human CFTR cDNA. As expected, studies with vectors coding for marker genes or for CFTR cDNA demonstrated that airway administration of a vector does not yield efficient gene transfer, if there has been prior recent airway administration of the same Ad subgroup. In contrast, effective expression from the second administration can be achieved with an adenovirus vector belonging to a subgroup different from the first adenovirus administered. These data support the paradigm of alternating Ad vectors derived from different subgroups as strategy to circumvent anti-Ad humoral immunity, thus permitting the use of Ad vectors as a means to treat the respiratory manifestations of CF.

Adenoviruses, Human↗
PubMed1998

New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses.

The presence of replication-competent adenoviruses (RCAs) in batches of replication-defective adenovirus (Ad) vectors is a major problem for the application of these vectors in gene therapy. RCAs are generated by recombination between sequences in the Ad vector and homologous Ad sequences in the helper cells, resulting in the acquisition by the vector of early region 1. To prevent the formation of RCAs, we have developed helper cell lines, which we named PER, and matched Ad vectors that do not have sequence overlap. PER cells contain the Ad serotype 5 (Ad5) E1A- and E1B-encoding sequences (Ad5 nucleotides 459-3510) under the control of the human phosphoglycerate kinase (PGK) promoter. We demonstrate that PER cells synthesize high levels of the Ad5 E1A and E1B proteins. The yields from PER cells of E1-deleted Ads are similar to those obtained from earlier helper cells, such as 911 and 293 cells. Propagation of matched Ad vectors, which lack Ad5 nucleotides 459-3510, in one of the PER clones, PER.C6, does not result in the generation of RCAs, in contrast to propagation in 293 cells. We conclude that the combination of PER.C6 cells and nonoverlapping E1-deleted adenoviral vectors eliminates the problem of RCA generation by homologous recombination, and allows cost-effective production of safe, clinical-grade batches of recombinant Ad vectors.

Adenovirus E1A Proteins↗
PubMed2007

Rapid construction of small interfering RNA-expressing adenoviral vectors on the basis of direct cloning of short hairpin RNA-coding DNAs.

In the conventional method for constructing an adenoviral (Ad) vector expressing small interfering RNA (siRNA), short hairpin RNA (shRNA)-coding oligonucleotides are introduced downstream of a polymerase III (or polymerase II)-based promoter cloned into a shuttle plasmid. An siRNA expression cassette, which is cloned into the shuttle plasmid, is then introduced into the E1 deletion region of the Ad vector plasmid by in vitro ligation or homologous recombination in Escherichia coli, and the linearized plasmid is transfected into 293 cells, generating an Ad vector expressing siRNA. Therefore, two-step plasmid manipulation is required. In this study, we developed a method by which shRNA-coding oligonucleotides can be introduced directly into the Ad vector plasmid. To do this, we constructed a new vector plasmid into which the human U6 promoter sequence was cloned in advance. Unique restriction enzyme sites were introduced at the transcription start site of the U6 promoter sequence in the vector plasmid. Luciferase and p53 genes were efficiently knocked down by Ad vectors generated by the new method and expressing siRNA against the target gene. This method should be useful for RNA interference-based experiments, and should make it easy to construct an siRNA-expressing Ad vector library for functional screening.

Adenoviridae↗
PubMed2006

Synthetic intron improves transduction efficiency of trans-splicing adeno-associated viral vectors.

Trans-splicing adeno-associated viral (AAV) vectors hold great promise in many gene therapy applications. We have shown that rational selection of the gene-splitting site in a therapeutic target gene can lead to extremely efficient trans-splicing vectors [Lai, Y., Yue, Y., Liu, M., Ghosh, A., Engelhardt, J.F., Chamberlain, J.S., and Duan, D. (2005). Nat. Biotechnol. 23, 1435-1439]. Our original strategy requires the screening of endogenous introns that are capable of overcoming the mRNA accumulation barrier. To further develop transsplicing vectors, we have tested whether the use of a generic synthetic intron can bypass the labor-intensive intron-screening process. Two previously characterized exon/intron/exon junctions (60/60/61 and 63/63/64, respectively) in the 6 kb minidystrophin gene were used as templates to represent highly efficient (60/60/61) and relatively poor (63/63/64) gene-splitting sites. We compared RNA production from the reconstituted viral genome and transduction efficiency of the trans-splicing vectors in dystrophin-null mdx mouse skeletal muscle. Our results suggest that a synthetic intron can successfully overcome the mRNA accumulation barrier at the exon 63/64 junction. Furthermore, when the gene was split at the exon 63/64 junction, the synthetic intronbased vectors performed better than the endogenous intron-based vectors. When the gene was split at the exon 60/61 junction, we observed only nominal improvement in mRNA production. Nevertheless, vectors based on the exon 60/61 junction remain the best set in transduction efficiency. Taken together, our results suggest that optimizing intron sequence may boost the transduction efficiency of trans-splicing AAV vectors.

Animals↗