Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.

Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0 ± 20.8%) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%), which is comparable with that of AAV sero-negative animals (53.7 ± 7.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.

Eye position influences auditory responses in primate inferior colliculus.

We examined the frame of reference of auditory responses in the inferior colliculus in monkeys fixating visual stimuli at different locations. Eye position modulated the level of auditory responses in 33% of the neurons we encountered, but it did not appear to shift their spatial tuning. The effect of eye position on auditory responses was substantial-comparable in magnitude to that of sound location. The eye position signal appeared to interact with the auditory responses in at least a partly multiplicative fashion. We conclude that the representation of sound location in primate IC is distributed and that the frame of reference is intermediate between head- and eye-centered coordinates. The information contained in these neurons appears to be sufficient for later neural stages to calculate the positions of sounds with respect to the eyes.

Redox regulation of vascular smooth muscle cell differentiation.

Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of differentiation proteins (alpha-actin, calponin, and SM1 and SM2 myosin), but not beta-actin. ROS activity, determined using the H(2)O(2)-sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A superoxide dismutase mimic (SOD; MnTMPyP, 25 micromol/L) or SOD plus low concentrations of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence, which was inhibited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity (by catalase or NAC) decreased the baseline expression of differentiation proteins, whereas elevation of ROS (by SOD or SODNAC2) increased expression of the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to 20 mmol/L). None of the treatments altered beta-actin expression. SODNAC2-treated cells demonstrated contractions to endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased when ROS activity was reduced (NAC, 10 mmol/L) and was augmented when ROS activity was increased (SODNAC2). Inhibition of p38 MAPK with pyridyl imidazole compound (SB202190, 2 to 10 micromol/L) reduced expression of differentiation proteins occurring under basal conditions and in response to SODNAC2. Transduction of VSMCs with an adenovirus encoding constitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the differentiation proteins. These findings demonstrate that ROS can increase VSMC differentiation through a p38 MAPK-dependent pathway.

Cell lines for the production of recombinant adeno-associated virus.

Adeno-associated virus (AAV) is a replication-defective parvovirus that is being developed as a vector for human gene transfer. However, a major obstacle to commonplace usage of AAV vectors is the production of recombinant virions (rAAV) in sufficient quantities for not only human trials, but also for preclinical studies of basic biology, toxicology, and efficacy. Unfortunately, current methods for large-scale production are cumbersome and expensive. We have developed a simplified method for generating rAAV by establishing neomycin-resistant cell lines containing copies of the AAV rep-cap genes and a rAAV vector. After infection with adenovirus, these cell lines are shown to produce infectious rAAV in relatively high titer. This method eliminates the need for exogenous DNA transfection and scale-up procedures are limited only by the normal constraints of growing cells in culture.

Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses.

Gene transfer vectors based on the replication-defective human parvovirus, adeno-associated virus type 2 (AAV-2), are viable candidates for in vivo and ex vivo human use. However, widespread testing of AAV vectors has been limited by difficulties in generating pure, high-titer vector stocks that are fully characterized. To address these issues, we have developed a single-step purification scheme using heparin affinity chromatography. Recovery from the crude lysate starting material exceeds 70%, and the end product rAAV vector is highly purified and appears to be free of adenovirus and cellular contaminates. Importantly, purified vectors retain predicted in vivo biologic activity. Concurrently, we have developed simple and rapid approaches for vector quantification using real-time PCR. These new methods, combined with the use of stable producer cell lines for rAAV production, make the commercial production of rAAV vectors for human use truly viable and pragmatic.

Recombinant adeno-associated viral vectors mediate long-term transgene expression in muscle.

Gene transfer to muscle holds overt promise for the treatment of inherited myopathies, lysosomal storage disorders, and serum protein deficiencies. In addition, muscle could provide a reservoir for delivery of therapeutic molecules like blood clotting factors, erythropoietin, or insulin. To date, successful gene transfer to muscle has been limited by the inefficiency of the vector delivery systems and the transient nature of gene expression. In this paper, we show that a vector based on recombinant adeno-associated virus (rAAV) can efficiently transduce adult mouse skeletal muscle. Transduced myofibers escape immune elimination and transgene expression is robust beyond 5 months. Importantly, input vector DNA appears to undergo conversion from single-stranded genomes to high-molecular-weight concatameric forms. These data suggest that rAAV might have a significant advantage over many other viral and nonviral gene delivery methods, and holds significant promise as a vector for gene transfer to mature muscle.

Recombinant adeno-associated virus-mediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type VII mouse.

The central nervous system (CNS) is a predominant site of involvement in several lysosomal storage diseases (LSDs); and for many patients, these diseases are diagnosed only after the onset of symptoms related to the progressive accumulation of macromolecules within lysosomes. The mucopolysaccharidosis type VII (MPS VII) mice are deficient for the lysosomal enzyme beta-glucuronidase and, by early adulthood, develop a significant degree of glycosaminoglycan storage within neuronal, glial, and leptomeningeal cells. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) vector is capable of reversing the progression of storage lesions within the CNS. Adult MPS VII mice received intracerebral injections of 4 X 10(7) infectious units of a rAAV vector carrying the murine beta-glucuronidase (gus-s(a)) cDNA under the transcriptional direction of the cytomegalovirus immediate-early promoter and enhancer. By 1 month after vector administration, transgene-derived beta-glucuronidase was present surrounding the injection site. Enzyme levels were between 50 and 240% of that found in wild-type mice. This level of beta-glucuronidase activity was sufficient to reduce the degree of lysosomal storage. Moreover, the reduction in storage was maintained for at least 3 months post-rAAV administration. These data demonstrate that rAAV vectors can transduce the diseased CNS of MPS VII mice and mediate levels of transgene expression necessary for a therapeutic response. Thus, rAAV vectors are potential tools in the treatment of the mucopolysaccharidoses and other lysosomal storage diseases.

Gene delivery of vaccines for infectious disease.

Genetic immunization is the process of delivering and expressing a gene (or therapeutic nucleic acid) encoding a pathogen-derived antigen into target host cells to elicit a protective humoral or cell-mediated immune response. Gene delivery methods to achieve this goal have expanded rapidly, and currently employ a variety of oligonucleotides, synthetic polypeptides, recombinant vectors and even edible plants, all of which have been shown to be capable of inducing protective immunity in experimental animal models. This review highlights recent progress in several gene delivery systems (both non-viral and viral methods) using novel in vivo approaches to engender effective host immune responses against the introduced antigen.

Breast abscess following central venous catheterization.

We describe a case of late perforation of the right internal mammary vein by a central venous catheter inserted via the left subclavian vein. This resulted in extravasation of parenteral nutritional fluid and breast abscess formation. Delay in reaching the diagnosis was experienced in this case and in two cases previously reported. The clinical presentation was similar in all three cases with good early function of the catheters followed by symptoms of chest pain and signs of inflammation in the breast contralateral to the site of insertion of the central venous catheter. Confirmation of correct placement of the central venous catheter must be sought in any patient who develops these symptoms or signs so that the delay in diagnosis we experienced can be avoided. This may be obtained by a lateral or oblique chest radiograph or by contrast studies if preferable.

Gene transfer into the CNS using recombinant adeno-associated virus: analysis of vector DNA forms resulting in sustained expression.

Recombinant adeno-associated virus (rAAV) vectors have shown significant promise as vehicles for in vivo gene transfer, particularly for transduction of organs composed primarily of non-dividing cells (i.e., muscle, CNS, and liver). However, the mechanistic basis for this desirable property remains unclear. To investigate the fate of rAAV genomes in mouse brain, we stereotactically injected an rAAV vector carrying the E. coli lacZ gene into the caudate of BALB/c mice and demonstrate efficient transduction of mouse brain cells that possess cellular morphology consistent with post-mitotic neurons. We observed a significant increase in beta-galactosidase expression from 5 to 56 days after injection that paralleled the disappearance of single-stranded DNA input genomes. Analysis of in vivo viral DNA forms over time out to 5 months after inoculation revealed that rAAV genomes associated with high molecular weight mouse chromosomal DNA by 14 days after injection and persisted for the length of this study. The pattern of Southern hybridization was consistent with random viral integration in predominantly head-to-tail concatameric arrays. Importantly, we also documented an additional DNA species that appears to be a monomeric episomal circular form based on nuclease sensitivity assays. These data are the first to document the existence of multiple vector DNA forms present within the adult murine brain following direct rAAV inoculation and therefore, provide insight into the molecular events that ultimately result in long-term rAAV mediated transgene expression.

Successful DNA transfer in cultured human mesangial cells using replication deficient recombinant adenovirus.

BACKGROUND: Efficient transfer of DNA into human mesangial cells is an essential first step in the development of gene therapies for mesangial cell-mediated glomerulopathies. In the present studies, we assessed the ability of replication deficient recombinant adenovirus to transfer DNA (transduce) into primary cultures of human mesangial cells. METHODS: Primary cultures of human mesangial cells were transduced with an adenoviral vector (rAv beta-gal) containing a CMVllacZ promoter-reporter expression cassette coding for beta-galactosidase (beta-gal). We assessed soluble and histologic beta-gal activity, morphology, and phenotypic expression of mesangial cell transductants, durability of transduced mesangial cells by measuring transgene expression following trypsinization or after prolonged periods in culture and metabolic stability following transduction (as assessed by fibronectin biosynthesis). RESULTS: We showed that rAv beta-gal efficiently transduced mesangial cells in a dose-dependent fashion at a multiplicity of infectious units (MOI) ranging from 1 to 400 plaque forming units/cell (pfu/cell). One hundred percent of mesangial cells were transduced at an MOI of 100 pfu/cell. By electron microscopic evaluation, viral particles of approximately 85-90 nm were demonstrated in the cytoplasm of transduced cells. Following transduction, legal levels rose rapidly and were 10-fold greater than baseline levels after 2 hours. Beta-gal levels continued to rise for 7 days following transduction. Transduction with rAv beta-gal was well tolerated; mesangial cell transductants maintained normal morphology and phenotype, tolerated 3 cycles of trypsinization and maintained normal constitutive production of fibronectin. CONCLUSIONS: Gene transfer with adenovirus is an effective, well tolerated approach for introducing DNA into primary cultures of human mesangial cells.

The effects of pedicle screw fit. An in vitro study.

STUDY DESIGN: The effects of pedicle screw size (major diameter and length) on fixation stiffness in osteoporotic and nonosteoporotic vertebrae were evaluated in vitro. METHODS: Lumbar vertebrae were obtained from two fresh frozen human spines. Bone mineral densities were determined using dual energy radiograph absorptiometry, followed by nondestructive mechanical testing of the specimens instrumented with pedicle screws. A loading technique was used that more closely mimics loading of pedicle screws in vivo. RESULTS: Testing revealed that for good quality bone, screw size had a significant effect on fixation stiffness, but the effect of penetration depth depended on pedicle fill, and vice versa. In nonosteoporotic bone, the use of a longer screw increased fixation stiffness if the screw filled up the pedicle by 70% or more. The use of wider screws increased the fixation stiffness if the penetration depth was 80% or more. CONCLUSION: Screw size had little or no effect on fixation stiffness in osteoporotic bone.

Surgical aspects of chronic peritoneal dialysis in the neonate and infant under 1 year of age.

Since 1982 eight patients under 1 year of age with end-stage renal failure have been treated by chronic peritoneal dialysis (CPD) following insertion of an abdominal Tenckhoff catheter. We routinely perform a partial omentectomy now, and in males undertake bilateral exploration of the groins at the time of catheter insertion, with herniotomy or ligation of the patent processus vaginalis as required. Up to January 1990, 19 straight double-cuff catheters had been inserted with a total follow-up of 244.5 patient months. The median age at the initial catheter insertion was 14.6 weeks (range, 2 days to 11 months) and the median weight was 3.89 kg (range, 2.2 to 5.5). Peritonitis was the most common complication, with 46 episodes, representing one episode of peritonitis per 5.3 patient months on dialysis. The frequency of peritonitis has decreased in the last 6 months since all patients have been dialysed by two caregivers. The present rate of peritonitis is 1 episode per 10 patient months on dialysis. One patient has died of septicemia secondary to associated congenital abnormalities, one patient has regained renal function, and two patients have been transplanted, one successfully. Five patients are currently dialysing via their abdominal Tenckhoff catheters and awaiting transplantation. We conclude that neonates and infants under 1 year of age can be treated satisfactorily by CPD to enable successful preparation for transplantation later in childhood.

Lack of toxicity of alpha-sarcoglycan overexpression supports clinical gene transfer trial in LGMD2D.

BACKGROUND: Alpha-sarcoglycan (alpha-SG) deficiency (limb-girdle muscular dystrophy [LGMD] type 2D) is the most common form of sarcoglycan-LGMD. No treatment is currently available. Prior studies suggest that overexpression of alpha-SG via adeno-associated virus (AAV)-mediated gene transfer results in poorly sustained gene expression related to transgene toxicity. These findings potentially preclude gene therapy as a treatment approach for LGMD2D. METHODS: The human alpha-SG gene (halpha-SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old alpha-SG KO mice using AAV, type 1. The gene was placed under control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK). Low (3 x 10(9) vg) and high (3 x 10(10) vg) doses of AAV1.halpha-SG were administered. RESULTS: Sustained gene expression was observed irrespective of promoters at 6 and 12 weeks post gene transfer. Quantitation of alpha-SG gene expression by fiber counts yielded similar levels of myofiber transduction for both MCK promoters (60 to 70%), while 34% of fibers were transduced with the DES promoter. There was a trend toward lower expression at the 12-week time point with the CMV promoter. Western blot analysis revealed alpha-SG overexpression using CMV and both the MCK promoters. CONCLUSION: Our data demonstrate robust and sustained adeno-associated virus type 1 alpha-sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of cytotoxicity. These findings support the use of gene therapy as a potential treatment approach for limb-girdle muscular dystrophy type 2D.