Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies

Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.     

Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo.

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.     

Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons.

Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAAVs is their limited capacity to package foreign DNA (< or =5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with cap-dependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-'cleavage' of the FMDV polyprotein in a cascade of processing events that ultimately generate the mature FMDV proteins. We have generated several different rAAV genomes in which two coding regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products from the artificial fusion proteins in cells. Furthermore, we find that both EGFP and alpha- synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while SOD-1 is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic gene expression in vivo after injection of 2A-dependent rAAVs into the rat substantia nigra. We conclude that 2A-containing rAAVs may represent an attractive alternative to IRES-dependent vectors for ex vivo and in vivo gene expression and gene therapy.     

Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat.

In vivo recombinant adeno-associated viral vector (rAAV)-mediated transduction of various tissues including brain has been characterized by slow onset and gradual increase in gene expression before reaching stable long-term protein levels. The early time course of transgene expression has not been quantified using newly available rAAV capsid serotypes. In this experiment, the onset of expression of green fluorescent protein (GFP) after intrastriatal injection of rAAV2-based pseudotyped vectors (rAAV1, rAAV5, and rAAV8 capsids) was quantified. Native GFP fluorescence displayed a delayed onset of expression of at least 7 days for all the pseudotyped rAAV vectors. However, GFP immunohistochemical staining revealed significant transgene expression by 4 days after transduction for all serotypes and stable GFP(+) neuronal populations mediated by all serotypes within 14 days post transduction at the latest. rAAV2/1 and rAAV2/2 displayed no time-dependent increase of GFP(+) striatal neurons; reaching maximal striatal cell GFP(+) counts at 4 days after injection. All serotypes displayed peak transgene expression by 4 weeks post injection where native GFP(+) neurons were equal to immunostained striatal GFP(+) neurons. The inflammatory response to these rAAV vectors was present up to 4 weeks after transduction but was not apparent 9 months post injection. Thus, rAAV-mediated transgene expression begins earlier than previously thought.     

Widespread and Efficient Transduction of Spinal Cord and Brain Following Neonatal AAV Injection and Potential Disease Modifying Effect in ALS Mice

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.     

Systemic mannitol-induced hyperosmolality amplifies rAAV2-mediated striatal transduction to a greater extent than local co-infusion.

Recombinant viral vectors derived from adeno-associated virus serotype 2 (rAAV2) have been investigated as highly effective vehicles for gene transfer to the central nervous system (CNS). Transduction with rAAV2 vectors results in long-term transgene expression in CNS neurons. Optimal injection parameters leading to efficient targeting and spread of the transgene to large neuroanatomical regions are important in molecular gene therapy studies of the CNS. In the present study, we reexamined the effects of both local and systemic administration of mannitol-induced hyperosmolality on facilitation of transgene expression and vector distribution in the CNS. Systemic intraperitoneal administration of mannitol prior to vector administration improved gene transfer to striatal neurons, increasing the total number of transduced cells by 400% and vector distribution by 200%. On the other hand, local coadministration of mannitol in the striatum increased the number of transduced striatal neurons by 25% and had little effect on transduction volume. In conclusion, we have demonstrated that systemic coadministration of mannitol significantly enhances transgene spread of rAAV2 viral delivery in the brain to a much greater degree than local coadministration.     

Neurological Correction of Lysosomal Storage in a Mucopolysaccharidosis IIIB Mouse Model by Adeno-associated Virus-Mediated Gene Delivery

Mucopolysaccharidosis (MPS) IIIB is characterized by mild somatic features and severe neurological diseases leading to premature death. No definite treatment is available for MPS IIIB patients. We constructed two recombinant adeno-associated virus (rAAV) vectors containing the human α-N-acetylglucosaminidase (NaGlu) cDNA driven by either a CMV or a neuron-specific enolase (NSE) promoter. In vitro, these rAAV vectors mediated efficient expression of recombinant NaGlu in human MPS IIIB fibroblasts and mouse MPS IIIB somatic and brain primary cell cultures. The secreted rNaGlu was taken up by both human and mouse MPS IIIB cells in culture and degraded the accumulated glycosaminoglycans (GAG). A direct microinjection (10⁷ viral particles, 1 μl/10 minutes per injection) of vectors containing the NSE promoter resulted in long-term (6 months, the duration of the experiments) expression of rNaGlu in multiple brain structures/areas of adult MPS IIIB mice. Consistent with previous studies, the main target cells were neurons. However, while vector typically transduced an area of 400–500 μm surrounding the infusion sites, the correction of GAG storage involved neurons of a much broader area (1.5 mm) in a 6-month duration of experiments. These results provide a basis for the development of a treatment for neurological disease in MPS IIIB patients using AAV vectors.     

Several rAAV Vectors Efficiently Cross the Blood–brain Barrier and Transduce Neurons and Astrocytes in the Neonatal Mouse Central Nervous System

Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood–brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.     

Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector

Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/beta-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.     

Recombinant adeno-associated virus vector-transduced vascular endothelial cells express the thrombomodulin transgene under the regulation of enhanced plasminogen activator inhibitor-1 promoter

We were able to facilitate plasminogen activator inhibitor 1 (PAI-1) promoter activity approximately by 14-fold using an enhancer element. This enhanced PAI-1 promoter has a strong basal activity, comparable to CAG promoter activity, and has a response similar to the PAI-1 promoter with respect to TGFbeta 1 and TNFalpha stimulation. The characteristics of the enhanced PAI-1 promoter are thought to be suited to timely and tissue-specific expression of anticoagulant molecules in the vascular cells. Thus, we developed recombinant adeno-associated virus (rAAV) vectors using the enhanced PAI-1 promoter and were successful in transducing vascular endothelial cells to express the thrombomodulin transgene under the regulation of the enhanced PAI-1 promoter in vitro. Thromobomodulin transgene expression driven by the enhanced PAI-1 promoter in rAAV vector-transduced cultured endothelial cells was between 600- and 1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGFbeta1 and TNFalpha stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils could also be transduced by the same rAAV vector in vivo. Transduction of endothelial cells by rAAV vectors to express enhanced PAI-1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.     

Gene therapy for Parkinson's disease using recombinant adeno-associated viral vectors

Existing strategies for gene therapy in the treatment of Parkinson's disease include the delivery of genes encoding dopamine (DA)-synthesising enzymes, leading to localised production of DA in the striatum; genes encoding factors that protect nigral neurons against ongoing degeneration, such as glial cell line-derived neurotrophic factor; and genes encoding proteins that produce the inhibitory transmitter gamma-aminobutylic acid (GABA) in the subthalamic nucleus (STN), thus suppressing the hyperactive STN. Recombinant adeno-associated viral (rAAV) vectors, which are derived from non-pathogenic viruses, have been shown to be suitable for clinical trials. These rAAVs have been found to transduce substantial numbers of neurons efficiently and to express transgenes in mammalian brains for long periods of time, with minimum inflammatory and immunological responses. In vivo imaging using positron emission tomography is useful for monitoring transgene expression and for assessing the functional effects of gene delivery. Vector systems that regulate transgene expression are necessary to increase safety in clinical applications, and the development of such systems is in progress.     

Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain

Recombinant adeno-associated virus serotype 2 (rAAV2) vectors have been shown to deliver genes effectively to neurons in the brain, retina, and spinal cord. The characterization of new AAV serotypes revealed different patterns of transduction in a diverse array of tissues (Gao, G., Vandenberghe, L.H., and Wilson, J.M. [2005]. Curr. Gene Ther. 5, 285-297). Here, we extensively compare the neural tropism of human-derived rAAVs (types 2/1, 2, and 2/5) with nonhuman primate-derived rAAVs (types 2/7 and 2/8) in adult mouse brain. Mice were injected with rAAV type 2/1, 2, 2/5, 2/7, or 2/8 via the caudate-putamen and substantia nigra. Intrahippocampal injections were also performed for rAAV2/7 and rAAV2/8. In all regions injected, the vectors transduced neurons almost exclusively. Retrograde transduction of all rAAV pseudotypes was also observed in particular CNS areas. At high titers, all rAAV pseudotypes transduced comparable brain volumes in all targeted regions except for rAAV2, which transduced much smaller brain volumes. A dose-range comparison of intrastriatally injected rAAV types 2/5, 2/7, and 2/8 highlighted that the transduction efficiency, as determined by transduced volume and biophotonic imaging of green fluorescent protein expression intensity, was significantly higher for rAAV2/5 and rAAV2/7 compared with rAAV2/8 at low titers, whereas all three serotypes performed equally well at higher doses. These results demonstrate the use and efficiency of both human- and nonhuman primate-derived rAAV vectors for disease modeling and their potential for gene therapy.     

Efficient and stable transduction of dopaminergic neurons in rat substantia nigra by rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9

Dysfunction of the nigrostriatal system is the major cause of Parkinson's disease (PD). This brain region is therefore an important target for gene delivery aiming at disease modeling and gene therapy. Recombinant adeno-associated viral (rAAV) vectors have been developed as efficient vehicles for gene transfer into the central nervous system. Recently, several serotypes have been described, with varying tropism for brain transduction. In light of the further development of a viral vector-mediated rat model for PD, we performed a comprehensive comparison of the transduction and tropism for dopaminergic neurons (DNs) in the adult Wistar rat substantia nigra (SN) of seven rAAV vector serotypes (rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9). All vectors were normalized by titer and volume, and stereotactically injected into the SN. Gene expression was assessed non-invasively and quantitatively in vivo by bioluminescence imaging at 2 and 5 weeks after injection, and was found to be stable over time. Immunohistochemistry at 6 weeks following injection revealed the most widespread enhanced green fluorescence protein expression and the highest number of positive nigral cells using rAAV 2/7, 2/9 and 2/1. The area transduced by rAAV 2/8 was smaller, but nevertheless almost equal numbers of nigral cells were targeted. Detailed confocal analysis revealed that serotype 2/7, 2/9, 2/1 and 2/8 transduced at least 70% of the DNs. In conclusion, these results show that various rAAV serotypes efficiently transduce nigral DNs, but significant differences in transgene expression pattern and level were observed.     

Characterization of Genome Integrity for Oversized Recombinant AAV Vector

Application of recombinant adeno-associated virus (rAAV) in gene therapy has been limited by its packaging capacity. Recent studies suggested that rAAV could achieve persistent transgene expression beyond 4.7-kb packaging limit. To clarify the mechanism leading to transgene expression from oversized rAAV vector, we constructed a series of rAAV vectors with genomes ranging from 2.9 to 7.2 kb. A plasmid replication origin and an ampicillin-resistant marker were included in the vector to facilitate the recovery of circularized, post-transduction AAV genome. Southern dot-blot analysis and silver staining confirmed that rAAVs could be produced at varying vector size. However, the vector yields decreased approximately tenfold for oversized vectors as compared to regular ones. Alkaline Southern blot hybridization suggested that the packaged genomes for oversized vectors were truncated. In the cells transduced by the above vectors, circularized rAAV monomers could be rescued at 24 hours after infection. Few recovered AAV genomes were >5 kb regardless of the initial vector size. In mice receiving the above vectors, larger circularized rAAV genomes could be recovered for oversized vectors at day 21 after vector administration. Our studies suggested that the partially packaged rAAV sequences may complement each other to restore full expression cassette.