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Biomedical subjects

R Jude Samulski

Publications and source records attributed to R Jude Samulski.

At least 19 recordsLinked to original sources
PubMed2017

Systemic and Persistent Muscle Gene Expression in Rhesus Monkeys with a Liver De-Targeted Adeno-Associated Virus Vector.

The liver is a major off-target organ in gene therapy approaches for cardiac and musculoskeletal disorders. Intravenous administration of most of the naturally occurring adeno-associated virus (AAV) strains invariably results in vector genome sequestration within the liver. In the current study, we compared the muscle tropism and transduction efficiency of a liver de-targeted AAV variant to AAV9 following systemic administration in newborn rhesus monkeys. In vivo bioluminescence imaging was performed to monitor transgene expression (firefly luciferase) post administration. Results indicated comparable and sustained levels of systemic firefly luciferase gene expression in skeletal muscle over a period of two years. Quantitation of vector biodistribution in harvested tissues post-administration revealed widespread recovery of vector genomes delivered by AAV9 but markedly decreased levels in major systemic organs from the AAV variant. These studies validate the translational potential and safety of liver de-targeted AAV strains for gene therapy of muscle-related diseases.

Animals↗
PubMed2011

Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

Amino Acid Sequence↗
PubMed2010

AAV's anatomy: roadmap for optimizing vectors for translational success.

Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.

Cystic Fibrosis↗
PubMed2009

Reducing the risk of adeno-associated virus (AAV) vector mobilization with AAV type 5 vectors.

Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

Animals↗
PubMed2006

Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes.

Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.

Amino Acid Substitution↗
PubMed2006

Adeno-associated virus serotypes: vector toolkit for human gene therapy.

Recombinant adeno-associated viral (AAV) vectors have rapidly advanced to the forefront of gene therapy in the past decade. The exponential progress of AAV-based vectors has been made possible by the isolation of several naturally occurring AAV serotypes and over 100 AAV variants from different animal species. These isolates are ideally suited to development into human gene therapy vectors due to their diverse tissue tropisms and potential to evade preexisting neutralizing antibodies against the common human AAV serotype 2. Despite their prolific application in several animal models of disease, the mechanisms underlying selective tropisms of AAV serotypes remain largely unknown. Efforts to understand cell surface receptor usage and intracellular trafficking pathways exploited by AAV continue to provide significant insight into the biology of AAV vectors. Such unique traits are thought to arise from differences in surface topology of the capsids of AAV serotypes and variants. In addition to the aforementioned naturally evolved AAV isolates, several strategies to engineer hybrid AAV serotype vectors have been formulated in recent years. The generation of mosaic or chimeric vectors through the transcapsidation or marker-rescue/domain-swapping approach, respectively, is notable in this regard. More recently, combinatorial strategies for engineering AAV vectors using error-prone PCR, DNA shuffling, and other molecular cloning techniques have been established. The latter library-based approaches can serve as powerful tools in the generation of low-immunogenic and cell/tissue type-specific AAV vectors for gene delivery. This review is focused on recent developments in the isolation of novel AAV serotypes and isolates, their production and purification, diverse tissue tropisms, mechanisms of cellular entry/trafficking, and capsid structure. Strategies for engineering hybrid AAV vectors derived from AAV serotypes and potential implications of the rapidly expanding AAV vector toolkit are discussed.

Animals↗
PubMed2006

Minimizing oxidative stress by gene delivery of superoxide dismutase accelerates regeneration after transplantation of reduced-size livers in the rat.

Transplantation of reduced-size livers may lead to a hypermetabolic state and increased production of oxygen radicals. Since oxygen radicals may cause liver injury and impair liver regeneration, we tested the hypothesis that overexpression of superoxide dismutase (SOD) in reduced-size livers (RSL) would accelerate regeneration and reduce injury in a rat model of transplantation of RSL. Donor rats were infected with adenoviruses either expressing SOD1 (Ad.SOD1) or beta-galactosidase (Ad.lacZ). Livers were harvested 72 hours later, reduced to 45% of weight, and transplanted. After transplantation, hepatic SOD activity, graft survival, histopathology, AST/ALT release, and bilirubin were examined. Regeneration was evaluated by BrdU-staining, graft weight, and expression of cyclin D1 and p21. In Ad.SOD1-treated livergrafts, SOD activity increased three-fold compared to controls. Survival was dramatically increased in recipients of Ad.SOD1-RSL (100% vs. 20% in Ad.lacZ-RSL), and peak levels of AST/ALT and bilirubin levels were reduced by 75% and 87.5%, respectively (P < 0.001). In histological sections, hepatocyte necrosis decreased from 24% after Ad.lacZ-treatment to 6% after Ad.SOD1-treatment (P <0.001). Regeneration was also accelerated after Ad.SOD1-treatment as demonstrated by an increase of BrdU-stained cells 24 hours after reperfusion and increased liver weight after 1 week. In conclusion, overexpression of SOD1 in RSL prevents primary non-function of reduced-size liver grafts and accelerates liver regeneration.

Adenoviridae↗
PubMed2006

Host cell DNA repair pathways in adeno-associated viral genome processing.

Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PK(CS), but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.

Animals↗
PubMed2005

Serotype-specific replicating AAV helper constructs increase recombinant AAV type 2 vector production.

One of the major limitations of the use of adeno-associated virus (AAV) as a tool for gene therapy is the difficulty in providing sufficient quantities of the virus for pre-clinical and clinical trials. Here, we report a novel approach for amplifying AAV helper functions, which mimics the normal replication of wild type (wt) AAV resulting in a high yield of AAV vectors. Cotransfection of replicating but non-packaging AAV helper constructs in the presence of adenovirus (Ad) produces a high level of Rep and Cap proteins. Yield of AAV2/GFP vector obtained from this helper DNA replication system was approximately 20-fold higher than traditional methods. Molecular analysis suggested that virus yield was associated with capsid protein production. The transfection ratio was optimized using these novel helper constructs, resulting in an additional 2-fold increase in vector yield without presence of replication competent AAV (rcAAV). This strategy supports development of AAV packaging systems that retain normal virus replication capability without helper virus encapsidation.

Cell Line↗
PubMed2005

Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy.

Structural allograft healing is limited because of a lack of vascularization and remodeling. To study this we developed a mouse model that recapitulates the clinical aspects of live autograft and processed allograft healing. Gene expression analyses showed that there is a substantial decrease in the genes encoding RANKL and VEGF during allograft healing. Loss-of-function studies showed that both factors are required for autograft healing. To determine whether addition of these signals could stimulate allograft vascularization and remodeling, we developed a new approach in which rAAV can be freeze-dried onto the cortical surface without losing infectivity. We show that combination rAAV-RANKL- and rAAV-VEGF-coated allografts show marked remodeling and vascularization, which leads to a new bone collar around the graft. In conclusion, we find that RANKL and VEGF are necessary and sufficient for efficient autograft remodeling and can be transferred using rAAV to revitalize structural allografts.

Animals↗
PubMed2005

Effects of adeno-associated virus DNA hairpin structure on recombination.

Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.

Aphidicolin↗
PubMed2005

AAV hybrid serotypes: improved vectors for gene delivery.

In recent years, significant efforts have been made on studying and engineering adeno-associated virus (AAV) capsid, in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest. With our previous knowledge of the viral properties of the naturally occurring serotypes and the elucidation of their capsid structures, we can now generate capsid mutants, or hybrid serotypes, by various methods and strategies. In this review, we summarize the studies performed on AAV retargeting, and categorize the available hybrid serotypes to date, based on the type of modification: 1) transcapsidation, 2) adsorption of bi-specific antibody to capsid surface, 3) mosaic capsid, and 4) chimeric capsid. Not only these hybrid serotypes could achieve high efficiency of gene delivery to a specific targeted cell type, which can be better-tailored for a particular clinical application, but also serve as a tool for studying AAV biology such as receptor binding, trafficking and genome delivery into the nucleus.

Animals↗
PubMed2004

Analysis of the interaction between adeno-associated virus and heparan sulfate using atomic force microscopy.

Adeno-associated virus (AAV) has been widely used as a viral vector to deliver genes to animal and human tissues in gene therapy studies. Both AAV-2 and AAV-3 use cell surface heparan sulfate (HS), a highly sulfated polysaccharide, as a receptor to establish infections. In this study, we used atomic force microscopy (AFM) to investigate the interaction of HS and AAV. A silicon chip functionalized with HS was used as a substrate for binding AAV for AFM analysis. To validate our approach, we found that the binding of AAV-2 to the HS surface was effectively competed by soluble HS, suggesting that the binding of AAV-2 to the functionalized surface was specific. In addition, we examined the binding of various AAV serotypes, including AAV-1, AAV-2, AAV-3, and AAV-5, to the HS surface. As expected, only AAV-2 and AAV-3 bound, whereas AAV-1 and AAV-5 did not. This observation was consistent with the previous conclusion that AAV-1 and AAV-5 do not use HS as a receptor for infection. In conclusion, we developed a novel approach to investigate the interaction of AAV virus with its polysaccharide-based receptor at the level of a single viral particle. Given that HSs serve as receptor for numerous viruses, this approach has the potential to become a generalized method for studying interactions between the viral particle and HS, as well as other virus-cell interactions, and potentially serve as a platform for screening antiviral therapies.

Cell Line↗
PubMed2004

Differential myocardial gene delivery by recombinant serotype-specific adeno-associated viral vectors.

Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors.

Animals↗
PubMed2004

Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups.

For all adeno-associated virus (AAV) serotypes, 60 monomers of the Vp1, Vp2, and Vp3 structural proteins assemble via an unknown mechanism to form an intact capsid. In an effort to better understand the properties of the capsid monomers and their role in viral entry and infection, we evaluated whether monomers from distinct serotypes can be mixed to form infectious particles with unique phenotypes. This transcapsidation approach consisted of the transfection of pairwise combinations of AAV serotype 1 to 5 helper plasmids to produce mosaic capsid recombinant AAV (rAAV). All ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these mixtures were able to replicate the green fluorescent protein transgene and to produce capsid proteins. A high-titer rAAV was obtained with mixtures that included either serotype 1, 2, or 3, whereas an rAAV of intermediate titer was obtained from serotype 5 mixtures. Only mixtures containing the AAV4 capsid exhibited reduced packaging capacity. The binding profiles of the mixed-virus preparations to either heparin sulfate (HS) or mucin agarose revealed that only AAV3-AAV5 mixtures at the 3:1 ratio exhibited duality in binding. All other mixtures displayed either an abrupt shift or a gradual alteration in the binding profile to the respective ligand upon increase of a capsid component that conferred either HS or mucin binding. The transduction of cell lines was used to further evaluate the phenotypes of these transcapsidated virions. Three transduction profiles were observed: (i) small to no change regardless of ratio, (ii) a gradual increase in transduction consistent with titration of a second capsid component, or (iii) an abrupt increase in transduction (threshold effect) dependent on the specific ratios used. Interestingly, an unexpected synergistic effect in transduction was observed when AAV1 helper constructs were combined with type 2 or type 3 recipient helpers. Further studies determined that at least two components contributed to this observed synergy: (i) heparin-mediated binding from AAV2 and (ii) an unidentified enhancement activity from AAV1 structural proteins. Using this procedure of mixing different AAV helper plasmids to generate "cross-dressed" AAV virions, we propose an additional means of classifying new AAV serotypes into subgroups based on functional approaches to analyze AAV capsid assembly, receptor-mediated binding, and virus trafficking. Exploitation of this approach in generating custom-designed AAV vectors should be of significant value to the field of gene therapy.

Animals↗
PubMed2004

Integration of adeno-associated virus (AAV) and recombinant AAV vectors.

The driving interest in adeno-associated virus (AAV) has been its potential as a gene delivery vector. The early observation that AAV can establish a latent infection by integrating into the host chromosome has been central to this interest. However, chromosomal integration is a two-edged sword, imparting on one hand the ability to maintain the therapeutic gene in progeny cells, and on the other hand, the risk of mutations that are deleterious to the host. A clearer understanding of the mechanism and efficiency of AAV integration, in terms of contributing viral and host-cell factors and circumstances, will provide a context in which to evaluate these potential benefits and risks. Research to date suggests that AAV integration in any context is inefficient, and that the persistence of AAV gene delivery vectors in tissues is largely attributable to episomal genomes.

Cell Line↗