Dataset Information

Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity.

ABSTRACT: The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (1 microl) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV-NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracyclineoff promoter (AAV-tTAK-GFP) and vector encoding beta-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector-derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy.

SUBMITTER: Haberman R

PROVIDER: S-EPMC3213639 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity.

Haberman Rebecca R Criswell Hugh H Snowdy Stephen S Ming Zhen Z Breese George G Samulski R R McCown Thomas T

Molecular therapy : the journal of the American Society of Gene Therapy 20021001 4

The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, ...[more]

PMID: 12377191

Similar Datasets

Project description:Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Project description:Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery.ImportanceAdeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy is met by intravitreal delivery, but delivery of AAV by this route results in poor transduction outcomes. The inner limiting membrane (ILM) creates a barrier separating the vitreous and the retina. Binding of AAV to heparan sulfate proteoglycan (HSPG) at the ILM may allow the virus to traverse this barrier for better retinal transduction. We show that HSPG binding is correlated with greater accumulation and penetration of AAV in the retina. We demonstrated that this accumulation is conserved across mouse and human retinas and that the addition of HSPG binding to other AAV capsids can increase the number of vectors accumulating at the ILM without dictating tropism.

Dataset Information

Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity.

Publications

Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets