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Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

Mucolipidoses II and III (ML II and ML III) are lysosomal disorders in which the mannose 6-phosphate recognition marker is absent from lysosomal hydrolases and other glycoproteins due to mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Both disorders are caused by the same gene, but ML II represents the more severe phenotype. Bone manifestations of ML II include hip dysplasia, scoliosis, rickets and osteogenesis imperfecta. In this study, we sought to determine whether a recombinant adeno-associated viral vector (AAV2/8-GNPTAB) could confer high and prolonged gene expression of GNPTAB and thereby influence the pathology in the cartilage and bone tissue of a GNPTAB knock out (KO) mouse model. The results demonstrated significant increases in bone mineral density and content in AAV2/8-GNPTAB-treated as compared to non-treated KO mice. We also showed that IL-6 (interleukin-6) expression in articular cartilage was reduced in AAV2/8-GNPTAB treated ML II mice. Together, these data suggest that AAV-mediated expression of GNPTAB in ML II mice can attenuate bone loss via inhibition of IL-6 production. This study emphasizes the value of the MLII KO mouse to recapitulate the clinical manifestations of the disease and highlights its amenability to therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.

Spinal muscular atrophy is a progressive motor neuron disease caused by a deficiency of survival motor neuron. In this study, we evaluated the efficacy of intravenous administration of a recombinant adeno-associated virus (AAV1) vector encoding human insulin-like growth factor-1 (IGF-1) in a severe mouse model of spinal muscular atrophy. Measurable quantities of human IGF-1 transcripts and protein were detected in the liver (up to 3 months postinjection) and in the serum indicating that IGF-1 was secreted from the liver into systemic circulation. Spinal muscular atrophy mice administered AAV1-IGF-1 on postnatal day 1 exhibited a lower extent of motor neuron degeneration, cardiac and muscle atrophy as well as a greater extent of innervation at the neuromuscular junctions compared to untreated controls at day 8 posttreatment. Importantly, treatment with AAV1-IGF-1 prolonged the animals' lifespan, increased their body weights and improved their motor coordination. Quantitative polymerase chain reaction and western blot analyses showed that AAV1-mediated expression of IGF-1 led to an increase in survival motor neuron transcript and protein levels in the spinal cord, brain, muscles, and heart. These data indicate that systemically delivered AAV1-IGF-1 can correct several of the biochemical and behavioral deficits in spinal muscular atrophy mice through increasing tissue levels of survival motor neuron.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). Previously, we showed that central nervous system (CNS) delivery of an adeno-associated viral (AAV) vector encoding SMN1 produced significant improvements in survival in a mouse model of SMA. Here, we performed a dose-response study in SMA mice to determine the levels of SMN in the spinal cord necessary for efficacy, and measured the efficiency of motor neuron transduction in the spinal cord after intrathecal delivery in pigs and nonhuman primates (NHPs). CNS injections of 5e10, 1e10, and 1e9 genome copies (gc) of self-complementary AAV9 (scAAV9)-hSMN1 into SMA mice extended their survival from 17 to 153, 70, and 18 days, respectively. Spinal cords treated with 5e10, 1e10, and 1e9 gc showed that 70-170%, 30-100%, and 10-20% of wild-type levels of SMN were attained, respectively. Furthermore, detectable SMN expression in a minimum of 30% motor neurons correlated with efficacy. A comprehensive analysis showed that intrathecal delivery of 2.5e13 gc of scAAV9-GFP transduced 25-75% of the spinal cord motor neurons in NHPs. Thus, the extent of gene expression in motor neurons necessary to confer efficacy in SMA mice could be obtained in large-animal models, justifying the continual development of gene therapy for SMA.

The GM2 gangliosidoses are progressive neurodegenerative disorders due to defects in the lysosomal β-N-acetylhexosaminidase system. Accumulation of β-hexosaminidases A and B substrates is presumed to cause this fatal condition. An authentic mouse model of Sandhoff disease (SD) with pathological characteristics resembling those noted in infantile GM2 gangliosidosis has been described. We have shown that expression of β-hexosaminidase by intracranial delivery of recombinant adeno-associated viral vectors to young adult SD mice can prevent many features of the disease and extends lifespan. To investigate the nature of the neurological injury in GM2 gangliosidosis and the extent of its reversibility, we have examined the evolution of disease in the SD mouse; we have moreover explored the effects of gene transfer delivered at key times during the course of the illness. Here we report greatly increased survival only when the therapeutic genes are expressed either before the disease is apparent or during its early manifestations. However, irrespective of when treatment was administered, widespread and abundant expression of β-hexosaminidase with consequent clearance of glycoconjugates, α-synuclein and ubiquitinated proteins, and abrogation of inflammatory responses and neuronal loss was observed. We also show that defects in myelination occur in early life and cannot be easily resolved when treatment is given to the adult brain. These results indicate that there is a limited temporal opportunity in which function and survival can be improved-but regardless of resolution of the cardinal pathological features of GM2 gangliosidosis, a point is reached when functional deterioration and death cannot be prevented.

Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezomib efficiently reduces AAV-specific IgG titres and moderates the cytotoxic T cell response in mice that have a pre-existing immunity to AAV2/8. Significant depletion of AAV2/8-specific IgG-producing plasma cells in secondary lymphoid organs and bone marrow was observed. However, this inhibition of the immune response by bortezomib was insufficient to allow subsequent re-infection with a recombinant AAV vector of a similar serotype. We show that this shortcoming is probably due to the combination of residual antibody levels and the inability of bortezomib to completely deplete the memory B cells that are re-activated in response to a repeated infection with a recombinant AAV vector. Taken together, the results of this study argue for the use of immunosuppressive therapies that target both plasma and memory B cells for the efficient elimination of pre-existing immunity against AAV2/8 vectors.

Sandhoff disease, a GM2 gangliosidosis caused by a deficiency in β-hexosaminidase, is characterized by progressive neurodegeneration. Although loss of neurons in association with lysosomal storage of glycosphingolipids occurs in patients with this disease, the molecular pathways that lead to the accompanying neurological defects are unclear. Using an authentic murine model of GM2 gangliosidosis, we examined the pattern of neuronal loss in the central nervous system and investigated the effects of gene transfer using recombinant adeno-associated viral vectors expressing β-hexosaminidase subunits (rAAV2/1-Hex). In 4-month-old Sandhoff mice with neurological deficits, cells staining positively for the apoptotic signature in the TUNEL reaction were found in the ventroposterior medial and ventroposterior lateral (VPM/VPL) nuclei of the thalamus. There was progressive loss of neuronal density in this region with age. Comparable loss of neuronal density was identified in the lateral vestibular nucleus of the brainstem and a small but statistically significant loss was present in the ventral spinal cord. Loss of neurons was not detected in other regions that were analysed. Administration of rAAV2/1-Hex into the brain of Sandhoff mice prevented the decline in neuronal density in the VPM/VPL. Preservation of neurons in the VPM/VPL was variable at the humane endpoint in treated animals, but correlated directly with increased lifespan. Loss of neurons was localized to only a few regions in the Sandhoff brain and was prevented by rAAV-mediated transfer of β-hexosaminidase gene function at considerable distances from the site of vector administration.

In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.

Several diseases of the nervous system are characterized by neurodegeneration and death in childhood. Conventional medicine is ineffective, but fetal or neonatal gene therapy may provide an alternative route to treatment. We evaluated the ability of single-stranded and self-complementary adeno-associated virus pseudotype 2/9 (AAV2/9) to transduce the nervous system and target gene expression to specific neural cell types following intravenous injection into fetal and neonatal mice, using control uninjected age-matched mice. Fetal and neonatal administration produced global delivery to the central (brain, spinal cord, and all layers of the retina) and peripheral (myenteric plexus and innervating nerves) nervous system but with different expression profiles within the brain; fetal and neonatal administration resulted in expression in neurons and protoplasmic astrocytes, respectively. Neither single-stranded nor self-complementary AAV2/9 triggered a microglia-mediated immune response following either administration. In summary, intravenous AAV2/9 targets gene expression to specific neural cell types dependent on developmental stage. This represents a powerful tool for studying nervous system development and disease. Furthermore, it may provide a therapeutic strategy for treatment of early lethal genetic diseases, such as Gaucher disease, and for disabling neuropathies, such as preterm brain injury.

Acute intermittent porphyria (AIP), an autosomal dominant hepatic porphyria due to half-normal hydroxymethylbilane synthase (HMB-synthase) activity, is manifested by life-threatening acute neurological attacks that are precipitated by factors that induce heme biosynthesis. The acute attacks are currently treated with intravenous hemin, but a more continuous therapy is needed, particularly for patients experiencing frequent attacks. Thus, a recombinant AAV8-based serotype vector expressing murine HMB-synthase driven by liver-specific regulatory elements was generated and its effectiveness to prevent the biochemical induction of an acute attack was evaluated in an AIP mouse model. Intraperitoneal administration of the adeno-associated viral (AAV) vector resulted in a rapid and dose-dependent increase of HMB-synthase activity that was restricted to the liver. Stable expression of hepatic HMB-synthase was achieved and wild-type or greater levels were sustained for 36 weeks. When heme synthesis was periodically induced by a series of phenobarbital injections, the treated mice did not accumulate urinary delta-aminolevulinic acid (ALA) or porphobilinogen (PBG), indicating that the expressed enzyme was functional in vivo and prevented induction of the acute attack. Further, rotarod performance and footprint analyses improved significantly. Thus, liver-directed gene therapy provided successful long-term correction of the hepatic metabolic abnormalities and improved neuromotor function in the murine model of human AIP.

Proteasome inhibitors have been shown to increase adeno-associated virus (AAV)-mediated transduction in vitro and in vivo. To assess if proteasome inhibitors also increase lipid-mediated gene transfer with relevance to cystic fibrosis (CF), we first assessed the effects of doxorubicin and N-acetyl-l-leucinyl-l-leucinal-l-norleucinal in non-CF (A549) and CF (CFTE29o-) airway epithelial cell lines. CFTE29o- cells did not show a response to Dox or LLnL; however, gene transfer in A549 cells increased in a dose-related fashion (p < 0.05), up to approximately 20-fold respectively at the optimal dose (no treatment: 9.3 x 10(4) +/- 1.5 x 10(3), Dox: 1.6 x 10(6)+/-2.6 x 10(5), LLnL: 1.9 x 10(6) +/- 3.2 x 10(5)RLU/mg protein). As Dox is used clinically in cancer chemotherapy we next assessed the effect of this drug on non-viral lung gene transfer in vivo. CF knockout mice were injected intraperitoneally (IP) with Dox (25-100 mg/kg) immediately before nebulisation with plasmid DNA carrying a luciferase reporter gene under the control of a CMV promoter/enhancer (pCIKLux) complexed to the cationic lipid GL67A. Dox also significantly (p < 0.05) increased expression of a plasmid regulated by an elongation factor 1alpha promoter (hCEFI) approximately 8-fold. Although administration of Dox before lung gene transfer may not be a clinically viable option, understanding how Dox increases lung gene expression may help to shed light on intracellular bottle-necks to gene transfer, and may help to identify other adjuncts that may be more appropriate for use in man.

The availability of a murine model of Pompe disease has enabled an evaluation of the relative merits of various therapeutic paradigms, including gene therapy. We report here that administration of a recombinant adeno-associated virus serotype 8 (AAV8) vector (AAV8/DC190-GAA) encoding human acid alpha-glucosidase (GAA) into presymptomatic Pompe mice resulted in nearly complete correction of the lysosomal storage of glycogen in all the affected muscles. A relatively high dose of AAV8/DC190-GAA was necessary to attain a threshold level of GAA for inducing immunotolerance to the expressed enzyme and for correction of muscle function, coordination, and strength. Administration of AAV8/DC190-GAA into older Pompe mice with overt disease manifestations was also effective at correcting the lysosomal storage abnormality. However, these older mice exhibited only marginal improvements in motor function and no improvement in muscle strength. Examination of histologic sections showed evidence of skeletal muscle degeneration and fibrosis in aged Pompe mice whose symptoms were abated or rescued by early but not late treatment with AAV8/DC190-GAA. These results suggest that AAV8-mediated hepatic expression of GAA was effective at addressing the biochemical and functional deficits in Pompe mice. However, early therapeutic intervention is required to maintain significant muscle function and should be an important consideration in the management and treatment of Pompe disease.

Background: Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Affected individuals can experience episodic, life-threatening, acute neurological attacks that are precipitated by various drugs, dieting, and hormonal changes. Intravenous hematin is used to treat the attacks, but a more effective, preventive therapy is needed, especially for patients with frequent attacks. Since the disease is a hepatic encephalopathy, efforts were focused towards evaluating four different combinations of liver-specific enhancers and promoters for maximal hepatic HMB-synthase expression., Methods: Four different mammalian expression vectors, each carrying a unique combination of liver-specific enhancers and promoters driving murine HMB-synthase cDNA expression, were transiently transfected into HepG2 cells. The vectors included: HMBS-1; human alpha1-microglobulin enhancer/alpha1-antityrpsin promoter (alpha1Me/alpha1ATp), HMBS-2; alpha1Me/human serum albumin promoter (alpha1Me/SAp), HMBS-3; human prothrombin enhancer/SAp (PTe/SAp), and HMBS-4; (PTe/alpha1ATp). Each HMB-synthase construct and a luciferase reporter construct were hydrodynamically coinjected into mice with HMB-synthase deficiency and evaluated for hepatic expression 24 h post-injection, the time-point of peak hepatic HMB-synthase expression., Results: Following transient transfection into HepG2 cells, HMBS-1 (alpha1Me/alpha1ATp) had the highest HMB-synthase expression level, with an approximately 8-fold increase over endogenous cellular activities. Construct HMBS-1 also had the highest hepatic HMB-synthase activity following hydrodynamic delivery into HMB-synthase deficient mice, with a approximately 6-fold increase over saline-treated mice., Conclusions: These studies support the use of a gene therapy vector containing the alpha1Me/alpha1ATp combination for preclinical studies of the efficacy and safety of liver-targeted gene therapy for AIP.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

Lysosomal storage disorders (LSD) are a group of approximately 40 genetic diseases that are caused by the deficiency of one or more lysosomal enzymes. The incidence of LSD is estimated to be approximately 1 in 7500 live births, which makes this one of the more prevalent groups of genetic diseases in humans. The loss in enzymatic activity leads to the accumulation of undegraded substrates within lysosomes, resulting in distension of the organelle and subsequent cellular malfunction. Although palliative treatments such as enzyme replacement therapy (ERT) or substrate reduction therapy (SRT) have been shown to be effective for some of the LSD such as Gaucher, Fabry and MPS I, they are not available as yet, or ineffective, for a large number of other LSD patients. To fulfill this unmet medical need, gene therapy is being considered as an alternate or adjunctive therapy for this group of disorders. A goal of gene therapy for LSD is to introduce a normal copy of the DNA for the lysosomal enzyme into a depot organ such as the liver or muscle with the intent that this will lead to the sustained production and re-constitution of therapeutic levels of the enzyme in the affected tissues. Here, we review the utility of various gene therapy strategies under consideration for the treatment of the LSD, including viral and non-viral gene transfer approaches, as well as stem cell transplantation.

Acid sphingomyelinase deficiency is a lysosomal storage disorder in which the defective lysosomal hydrolase fails to degrade sphingomyelin. The resulting accumulation of substrate in the lysosomes of histiocytic cells leads to hepatosplenomegaly and severe pulmonary inflammation. Administration of a recombinant AAV1 vector encoding human acid sphingomyelinase to acid sphingomyelinase knockout (ASMKO) mice effectively reduced the accumulated substrate in all of the affected visceral organs. However, more complete and rapid clearance of sphingomyelin was observed when an AAV8-based serotype vector was used in lieu of AAV1. Importantly, AAV8-mediated hepatic expression of higher and sustained levels of the enzyme also corrected the abnormal cellularity, cell differentials, and levels of the chemokine MIP-1alpha in the bronchoalveolar lavage fluids of the ASMKO mice. Treatment also reversed the morphological aberrations associated with the alveolar macrophages of ASMKO mice and restored their phagocytic activity. No antibodies to the expressed enzyme were detected when the viral vectors were used in conjunction with a transcription cassette harboring a liver-restricted enhancer/promoter. Together, these data support the continued development of AAV8-mediated hepatic gene transfer as an approach to treat the visceral manifestations observed in individuals with acid sphingomyelinase deficiency.

The mammalian innate immune system has the ability to recognise and direct a response against incoming foreign DNA. The primary signal that triggers this response is unmethylated CpG motifs present in the DNA sequence of various disease-causing pathogens. These motifs are rare in vertebrate DNA, but abundant in bacterial and some viral DNAs. Because gene therapy generally involves the delivery of DNA from either plasmids of bacterial origin or recombinant viruses, an acute inflammatory response of variable severity inevitably results. The response is most serious for non-viral gene delivery vectors composed of cationic lipid-DNA complexes, producing adverse effects at lower doses and lethality at higher doses of complex. This review examines the role of immunostimulatory CpG motifs in the acute inflammatory response to non-viral gene therapy vectors. Strategies to neutralise or eliminate CpG motifs within plasmid DNA vectors, and the existing limitations of CpG reduction on improving the safety profile of non-viral vectors, will be discussed.

Background: Previous studies have shown that incubation of balloon-injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the beta-adrenergic receptor kinase (Ad2/betaARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of betaARKct could be achieved using a catheter-based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis., Methods: In this study, Ad2/betaARKct was infused into the balloon-injured site of rabbit iliac arteries using a porous infusion catheter over 2 min. Twenty-eight days after gene transfer, angiographic and histological assessments were performed., Results: Angiographic and histological assessments indicate significant (p < 0.05) inhibition of iliac artery neointima formation and lumen stenosis by Ad2/betaARKct. Our studies demonstrate that an inhibitory effect of Ad2/betaARKct on neointima formation is achievable using a catheter-based vector delivery system and that the inhibition of neointima formation translates into a gain in the vessel minimal luminal diameter. The extent of inhibition (35%) was comparable to that observed with adenoviral-mediated expression of thymidine kinase plus ganciclovir treatment, a cytotoxic gene therapy approach for restenosis., Conclusions: These results suggest that adenoviral-mediated gene transfer of betaARKct is a clinically viable cytostatic gene therapy strategy for the treatment of restenosis., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

Fas, a member of the tumor necrosis factor receptor super-family, is expressed in all cell types examined, while physiologic expression of Fas ligand (FasL) is found predominantly in activated T-lymphocytes, vascular endothelial cells, and "immune-privileged" tissues. Activation of Fas following FasL binding activates caspases, which results in apoptosis. In the vasculature, there may be a delicate balance between cell proliferation and apoptosis in vascular smooth muscle cells. Shifts in this balance could account for the accumulation of vascular smooth muscle cells in response to arterial injury, a major feature of vascular intimal hyperplasia. Intimal hyperplasia occurs in more than a third of patients receiving percutaneous transluminal balloon angioplasty. Stenting with or without coating significantly reduces the incidence rate of angiographic restenosis and that of target vessel revascularization. However, "in-stent" intimal hyperplasia/restenosis remains a challenge for clinical cardiologists. Although both the cell types and mechanisms that contribute to intimal hyperplasia in response to vascular injury remain controversial, vascular smooth muscle cell migration and proliferation appear to play an important role in the process. In animal models, cytotoxic and cytostatic gene therapy strategies targeted at the vascular smooth muscle cells have shown therapeutic potential for the treatment of vascular intimal hyperplasia. However, Fas ligand-based gene therapy appears to offer several advantages. In this review article, we will discuss the mode of FasL/Fas signaling in vascular smooth muscle cells and its therapeutic implications. We will also compare the relative merits of FasL with other cytotoxic and cytostatic gene therapy approaches for the treatment of intimal hyperplasia.

Immunostimulatory CpG motifs have been implicated as a major contributor to the acute inflammatory response associated with nonviral vectors, most prominently seen after systemic delivery of cationic lipid-plasmid DNA (pDNA) complexes. We have shown previously that complexes containing pDNA vectors that have been largely depleted of CpG motifs have significantly reduced acute toxicity when delivered systemically. However, several CpGs remain in these vectors and the toxicity is not negligible, especially at higher doses of complex. To determine the maximal reduction in the acute toxic response that could be achieved by eliminating CpG signaling, we injected cationic lipid-pDNA complexes into transgenic mice that are deficient in Toll-like receptor 9 (TLR9), which is the receptor that recognizes immunostimulatory CpG motifs. We observed significantly decreased adverse hematological changes and liver damage in TLR9(-/-) mice compared to normal mice and increased survival at higher doses of complex. However, a pronounced loss of lymphocytes and platelets was still observed in the TLR9(-/-) mice at higher doses. We also measured the toxicity in normal mice of systemically delivered complexes containing non-CpG oligonucleotides. Although serum transaminase levels were reduced, a loss of lymphocytes and platelets akin to that seen in the TLR9(-/-) mice was observed. Taken together, these findings suggest that signaling through TLR9 contributes to the majority but not all of the toxic responses associated with systemic delivery of cationic lipid-pDNA complexes.

Background: Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A, leading to an accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in most tissues of the body. The goal of this study was to determine if systemic delivery of a nonviral vector could correct the enzyme deficiency and reduce the levels of GL-3 in different tissues of a transgenic knockout mouse model of the disease., Methods: Cationic lipid was complexed with a CpG-depleted plasmid DNA vector and then injected intravenously into Fabry mice. The levels of alpha-galactosidase A and GL-3 in different tissues were assayed at various time points after injection., Results: Expression of alpha-galactosidase A was detected in the different tissues of Fabry mice for up to 3 months after complex administration, but resulted in minimal reductions in GL-3 levels. However, the use of the anti-inflammatory drug dexamethasone and multiple dosing increased alpha-galactosidase A expression and resulted in significant reductions of GL-3 in all the organs with the exception of the kidney. In addition, injecting complex into young Fabry mice partially prevented the normal accumulation of GL-3 in the heart, lung, and liver., Conclusions: Systemic delivery of a cationic lipid-pDNA complex partially corrected the enzyme deficiency and reduced glycolipid storage in a mouse model of Fabry disease. The results are one of the few demonstrations of long-term efficacy in a genetic disease model using nonviral vectors. However, substantial improvements in expression, especially in critical organs such as the kidney, are required before these vectors can become a viable approach to treat Fabry disease and other lysosomal storage disorders., (Copyright 2003 John Wiley & Sons, Ltd.)

Systemic delivery of cationic lipid-plasmid DNA (pDNA) complexes induces an acute inflammatory response with adverse hematologic changes and liver damage. Immunostimulatory CpG motifs in the pDNA are known to contribute substantially to this response. Here we constructed a pDNA vector (pGZB) that has been depleted of 80% of the CpG motifs present in the original vector. Compared with the unmodified vector, systemic administration of pGZB induced considerably fewer changes in blood parameters, reduced levels of inflammatory cytokines, and decreased liver damage. Despite the extensive sequence modifications within pGZB, there were still robust levels of transgene expression. Furthermore, in contrast to the transient expression observed from the unmodified vector, we observed sustained or increasing expression for up to 49 days from pGZB in the lung and liver of immunocompetent BALB/c mice. Studies adding CpG motifs in trans or in cis indicate that the reduced CpG content of pGZB was the major determinant for its persistent expression. This combination of decreased toxicity and sustained expression suggests that CpG-depleted pDNA vectors can greatly improve the safety and efficacy of synthetic gene delivery systems., ((c)2002 Elsevier Science (USA).)

Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression. In Fabry mice, the reduced expression of alpha-galactosidase A, using the lower dose of Ad2/CMVHI-alpha gal, was associated with less than optimal clearance of the accumulated glycosphingolipid (GL-3) from the affected lysosomes. It was determined that this lack of linearity in dose response was not due to an inability to deliver the recombinant viral vectors to the liver but rather to sequestration, at least in part, of the viral vectors by the Kupffer cells. This lack of correlation between dose and expression levels could be obviated by supplementing the low dose of Ad2/CMVHI-alpha gal with an unrelated adenoviral vector or by depleting the Kupffer cells before administration of Ad2/CMVHI-alpha gal. Prior removal of the Kupffer cells, using clodronate liposomes, facilitated the use of a 100-fold lower dose of Ad2/CMVHI-alpha gal (10(9) particles/mouse) to effect the nearly complete clearance of GL-3 from the affected organs of Fabry mice. These results suggest that practical strategies that minimize the interaction between the recombinant adenoviral vectors and the reticuloendothelial system (RES) may improve the therapeutic window of this vector system. In this regard, we showed that pretreatment of mice with gamma globulins also resulted in significantly enhanced adenovirus-mediated transduction and expression of alpha-galactosidase A in the liver.

Niemann-Pick disease Type A (NPA) is a neuronopathic lysosomal storage disease (LSD) caused by the loss of acid sphingomyelinase (ASM). The goals of the current study are to ascertain the levels of human ASM that are efficacious in ASM knockout (ASMKO) mice, and determine whether these levels can be attained in non-human primates (NHPs) using a multiple parenchymal injection strategy. Intracranial injections of different doses of AAV1-hASM in ASMKO mice demonstrated that only a small amount of enzyme (10 mg hASM/g tissue was reached. In monkeys, injection of 12 tracts of AAV1-hASM resulted in efficacious levels of enzyme in broad regions of the brain that was aided, in part, by axonal transport of adeno-associated virus (AAV) and movement through the perivascular space. This study demonstrates that a combination cortical, subcortical, and cerebellar injection protocol could provide therapeutic levels of hASM to regions of the NHP brain that are highly affected in NPA patients. The information from this study might help design new AAV-mediated enzyme replacement protocols for NPA and other neuronopathic LSDs in future clinical trials.

The advent of novel adeno-associated virus (AAV) serotype vectors with higher transduction activity has encouraged a re-evaluation of the merits of this delivery platform for a variety of diseases. We report here that administration of a recombinant AAV8–based serotype vector encoding human α-galactosidase A into Fabry mice facilitated more rapid and significantly higher levels of production of the enzyme than an AAV2 vector. This translated into improved clearance of globotriaosylceramide, the glycosphingolipid that accumulates in the lysosomes of affected Fabry cells, and to correction of the peripheral neuropathy shown associated with this disease. The higher levels of α-galactosidase A expression also allowed for a more rapid induction of immunotolerance to the enzyme. Recombinant AAV8 vectors that facilitated hepatic–restricted expression of high levels of α-galactosidase A conferred immunotolerance to the expressed enzyme as early as 30 days post-treatment. Animals expressing lower levels of the hydrolase, such as those treated with an AAV2–based vector or with lower doses of the AAV8–based vector, were also able to develop immunotolerance, but only after a more extended time period. Adoptive transfer of T cells isolated from the spleens of immunotolerized mice suppressed the formation of antibodies in naïve recipient animals, suggesting the possible role of regulatory T cells in effecting this state.Molecular Therapy (2007) 15, 492–500. doi:10.1038/sj.mt.6300066; published online 26 December 2006 [ABSTRACT FROM AUTHOR]

The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human α-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-αgal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-αgal). Moreover, AAV2/DC190-αgal-mediated hepatic expression of α-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-αgal-treated animals with recombinant human α-galactosidase A at 6 months failed to elicit the production of anti-α-galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-αgal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of α-galactosidase A to the liver may be a viable strategy for treating Fabry disease. [Copyright &y& Elsevier]

We defined, using a novel large animal model system, the acute pathologic response to localized pulmonary administration of either naked plasmid DNA (pDNA) or cationic lipid–pDNA complexes (pDNA:GL67) and related such responses to concomitant indicators of transfection efficiency, namely levels of chloramphenicol acetyl transferase (CAT) protein and mRNA in specific lung tissue compartments. We instilled doses of 0.2, 1, and 5 mg pDNA to spatially distinct lung segments in six anesthetized sheep and doses of 0.2, 1, and 5 mg pDNA:GL67 to a further six sheep. Twenty-four hours after gene delivery the sheep were euthanized and necropsy examination with sampling of relevant tissues was carried out. Levels of plasmid-derived CAT-specific mRNA and CAT protein in samples derived from segments treated with either pDNA or pDNA:GL67 increased in relation to the administered dose. Levels of mRNA and protein expression were greater for pDNA:GL67 than for pDNA alone. A significant correlation was observed between mRNA and protein expression in samples derived from airways treated with pDNA:GL67. Histopathological changes following administration of both pDNA and pDNA:GL67 were characterized by a neutrophilic inflammation predominantly oriented on airways. The severity of the inflammatory response appeared to correlate with the administered dose of DNA and was generally more severe for pDNA:GL67. [Copyright &y& Elsevier]

Amytrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by a selective loss of motor neurons in the motor cortex, brain stem and spinal cord. The etiology of motor neuron degeneration remains largely unknown. Although chronic delivery of therapeutic agents to the central nervous system has proven to be difficult, recent studies have demonstrated that intramuscular delivery of adeno-associated viral vectors encoding insulin growth factor 1 (AAV-IGF-1) slowed disease progression and extended survival in SOD1G93A mice. The efficacy of viral mediated expression of IGF-1 was mediated in part by the ability of the viral vector to undergo retrograde transport from muscle to motor neurons. Previously we demonstrated that targeting the deep cerebellar nuclei (DCN) of the cerebellum with viral vectors capable of axonal transport resulted in detection of transgene protein in both the brain stem and spinal cord (Dodge et al. 2005, PNAS 102:17822-17827). In this experiment we evaluated the efficacy of bilateral delivery of AAV1–IGF-1 and AAV2-IGF-1 to the DCN in 90 day old SOD1G93A mice. Starting at 80 days of age (and every 10 days thereafter), SOD1G93A mice underwent behavioral testing (rotarod, hindlimb and forelimb grip strength) to assess motor function. At 90 days of age (i.e., time point at which SOD1 mice exhibit overt disease symptoms of ALS) mice received stereotaxic bilateral injections of AAV1-IGF-I (n=26), AAV2-IGF-I (n=27), AAV1-GFP (n=26) or AAV2-GFP (n=25) aimed at the deep cerebellar nuclei (DCN) of the cerebellum. We found that bilateral delivery of AAV- IGF-1 treatment (regardless of serotype) to the DCN significantly promoted motor neuron survival, improved motor performance in both rotarod and grip strength behavioral tests and significantly extended lifespan (i.e., 12 day extension for AAV1-IGF-1 and 14 day extension for AAV2-IGF-1). Treatment with AAV-GFP had no effect on any of the parameters mentioned above. Our results indicate that direct administration of AAV-IGF-1 into the DCN modifies disease progression in a mouse model of ALS and that targeting the DCN with AAV vectors is an effective approach for delivering therapeutic transgenes to both the brain and spinal cord for the treatment of ALS.Molecular Therapy (2006) 13, S407–S407; doi: 10.1016/j.ymthe.2006.08.1160 [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus serotypes-1 and -2 encoding human acid sphingomyelinase were administered into multiple structures of the cynomolgus monkey brain using convection- enhanced delivery. One group of monkeys (n = 2) was injected with 30–100 ul of AAV2-hASM into the striatum, thalamus, motor cortex and hippocampus, and euthanized at 31 days post-injection. Transduction occurred in the four injected structures and in other regions that resulted in an expression pattern that spanned 50–60 mm, although the posterior aspects of the brain had only sporadic human ASM-positive cells. In situ hybridization experiments demonstrated that widespread transduction was aided, in part, by retrograde axonal transport of AAV2 and by movement of the viral vector through the perivascular space. A second group of monkeys (n = 2) was injected with 30-50 ul of AAV1-hASM into the same four structures, as well as into the occipital cortex and cerebellum to achieve more efficient transduction of caudal brain structures. At 35 days post-injection, widespread transduction by AAV1 was observed in both the injected structures and in other regions along the rostro-caudal axis of the brain, including robust transduction of posterior aspect of the brain. In situ hybridization experiments confirmed retrograde axonal transport of AAV1 to multiple regions of the brain, including the medulla oblongata that sends axonal projections to the cerebellum. Furthermore, the horizontal transduction pattern in the neuronal layers of the convoluted cortex strongly suggests that AAV1 (and AAV2) may become widely dispersed by moving along the outside of major white matter tracts. This study demonstrate that the topographical organization of the brain circuits, and the anatomical features of the Virchow-Robin space and white matter tracts, may be exploited to achieve widespread gene/protein delivery in the primate brain including remote regions of that are not accessible by surgery.Molecular Therapy (2006) 13, S1–S1; doi: 10.1016/j.ymthe.2006.08.012 [ABSTRACT FROM AUTHOR]