![]() ![]() 1) to study the intermolecular trans-splicing production of Epo mRNA and protein. These proviral plasmids, termed pCisAV.Epo1 and pCisAV.Epo2, were used to produce recombinant viruses (Fig. By cutting at a unique BclI site within a central intron of the Epo gene, the Epo genomic locus was divided into two parts and inserted into two independent rAAV vectors by using pSub201 ( 11) as the source of inverted terminal repeats (ITRs). This fragment was inserted into the multiple cloning site of pIRES2-EGFP (CLONTECH) to generate pEpoEGFP, which expresses both Epo and enhanced green fluorescent protein (EGFP) from a single transcript. Materials and Methods Production of Recombinant Viral Vectors.Ī 2.4-kb BstEII/ BglII human genomic DNA fragment encoding the entire Epo gene was cloned from a lambda phage human genomic library (CLONTECH). These findings will greatly broaden the application of rAAV vectors for gene therapies to muscle, as well as to other organs and tissues having circular concatamerization as an inherent part of the rAAV life cycle. In this report, we demonstrate that this dual vector, trans-splicing strategy can result in the functional expression of therapeutic levels of protein from the erythropoietin (Epo) genomic locus. The design of this approach was based on the current understanding that rAAV vectors readily undergo a concatamerization process through intermolecular recombination from circular monomer intermediates of the viral genome ( 10). This approach entails the coadministration of two independent trans-splicing rAAV vectors encoding distinct segments of a large therapeutic gene and intron donor and acceptor signals. In this report, we describe a strategy permitting the delivery of large transgenes that exceed the normal packaging size of rAAV. ![]() Thus, diseases caused by defective genes larger than 5 kb, such as factor VIII deficiency or Duchenne muscular dystrophy, cannot presently be approached with this vector system. Circular intermediate formation and transgene persistence are intricately linked to a concatamerization process, which is controlled, at least in part, by intermolecular recombination between independent viral genomes ( 7, 10).ĭespite our increased understanding of rAAV transduction biology, the application of this vector in treating disease has been limited by the fundamental aspects of rAAV biology that define its packaging limits. Second, it has been shown that latent phase infection of muscle with rAAV involves the conversion of the single-stranded viral DNA to circular intermediate forms that correlate with long-term transgene expression ( 7– 9). First, the AAV-2 cell membrane receptor, heparan sulfate proteoglycan, as well as two coreceptors, fibroblast growth factor receptor-1 (FGFR-1) and α Vβ 5 integrin, have been identified ( 4– 6). Several important findings have recently emerged from studies in this area. ![]() Of foremost interest is the question why muscle tissue appears to be so tropic for this virus. With the promise of recombinant AAV (rAAV) vectors for muscle-directed therapies, a renewed interest in investigating the mechanisms of rAAV transduction in this tissue has emerged. These aspects include its potential for site-specific integration at the AAVS1 locus, its nonpathogenicity as a helper-dependent virus, and its ability to infect nondividing cells ( 1– 3). This virus has attracted considerable interest as a vector for gene therapy because of certain inherent aspects of its life cycle. These findings will allow for the application of AAV technologies to a wider variety of diseases for which therapeutic transgenes exceed the packaging limitation of present AAV vectors.Īdeno-associated virus (AAV) is a single-stranded DNA parvovirus with a helper-dependent life cycle. When two vectors encoding either the 5′ or 3′ portions of the erythropoietin genomic locus were used, functional erythropoietin protein was expressed in muscle subsequent to the formation of intermolecular circular concatamers in a head-to-tail orientation through trans-splicing between these two independent vector genomes. Based on the finding that AAV genomes undergo intermolecular circular concatamerization after transduction in muscle, we have developed a paradigm to increase the size of delivered transgenes with this vector through trans-splicing between two independent vectors coadministered to the same tissue. However, with a packaging size of <5 kb, applications have been limited to relatively small disease genes. Adeno-associated viral (AAV) vectors have demonstrated considerable promise for gene therapy of inherited diseases. ![]()
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