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Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.

ABSTRACT: Transduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 x 10(10) scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.

SUBMITTER: Nathwani AC

PROVIDER: S-EPMC1895379 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.

Nathwani Amit C AC Gray John T JT Ng Catherine Y C CY Zhou Junfang J Spence Yunyu Y Waddington Simon N SN Tuddenham Edward G D EG Kemball-Cook Geoffrey G McIntosh Jenny J Boon-Spijker Mariette M Mertens Koen K Davidoff Andrew M AM

Blood 20051201 7

Transduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-compleme ...[more]

PMID: 16322469

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Project description:Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes are greatly enhanced.

Dataset Information

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.

Publications

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.

Similar Datasets

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