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Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina.

ABSTRACT: Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after intravitreal injections, which differed according to the duration of diabetic induction. STZ was intraperitoneally injected into C57/B6 diabetic mice subjected to unilateral intravitreal injection of AAV2, AAV5, AAV8, and AAV9 packaged with EGFP. Significantly enhanced AAV2 and AAV9 transduction was observed in 2-month-old diabetic mouse retinas compared to the 2-week-old diabetic mouse retinas and nondiabetic, vector uninjected or injected retinas. Intravitreal injection of AAV5 or AAV8 serotype in 2-month- and 2-week-old diabetic mouse retinas did not show any significant vector transduction enhancement compared to the nondiabetic control retinas. The tropism of AAV2 and AAV9 in diabetic mouse retinas differed. AAV2 was transduced into various retinal cells, including Müller cells, microglia, retinal ganglion cells (RGCs), bipolar cells, horizontal cells, and amacrine cells, whereas AAV9 was effectively transduced only into RGC and horizontal cells. The expression levels of receptors and co-receptors for AAV2 and AAV9 were significantly increased in 2-month-old diabetic mouse retinas. The results of our study demonstrated that AAV2 and AAV9 may be the vector of choice in treating diabetic retinopathy (DR) with gene therapy, and DR-related retinal changes may improve AAV vector transduction efficiency.

SUBMITTER: Lee SH

PROVIDER: S-EPMC6330514 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina.

Lee Si Hyung SH Yang Jin Young JY Madrakhimov Sanjar S Park Ha Yan HY Park Keerang K Park Tae Kwann TK

Molecular therapy. Methods & clinical development 20181201

Adeno-associated viruses (AAVs) are currently the most popular vector platform technology for ocular gene therapy. While transduction efficiency and tropism of intravitreally administered AAV has been fairly well established in various retinal conditions, its transduction pattern in diabetic retinas has not previously been characterized. Here, we describe the transduction efficiencies of four different AAV serotypes, AAV2, 5, 8, and 9, in streptozotocin (STZ)-induced diabetic mouse retinas after ...[more]

PMID: 30666309

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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.

Project description:A better understanding of the molecular and cellular mechanisms involved in retinal hydro-mineral homeostasis imbalance during diabetic macular edema (DME) is needed to gain insights into retinal (patho-)physiology that will help elaborate innovative therapies with lower health care costs. Transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4) plays an intricate role in homeostatic processes that needs to be deciphered in normal and diabetic retina. Based on previous findings showing that TRPV4 antagonists resolve blood-retina barrier (BRB) breakdown in diabetic rats, we evaluated whether TRPV4 channel inhibition prevents and reverts retinal edema in streptozotocin(STZ)-induced diabetic mice. We assessed retinal edema using common metrics, including retinal morphology/thickness (histology) and BRB integrity (albumin-associated tracer), and also by quantifying water mobility through apparent diffusion coefficient (ADC) measures. ADC was measured by diffusion-weighted magnetic resonance imaging (DW-MRI), acquired ex vivo at 4 weeks after STZ injection in diabetes and control groups. DWI images were also used to assess retinal thickness. TRPV4 was genetically ablated or pharmacologically inhibited as follows: left eyes were used as vehicle control and right eyes were intravitreally injected with TRPV4-selective antagonist GSK2193874, 24 h before the end of the 4 weeks of diabetes. Histological data show that retinal thickness was similar in nondiabetic and diabetic wt groups but increased in diabetic Trpv4-/- mice. In contrast, DWI shows retinal thinning in diabetic wt mice that was absent in diabetic Trpv4-/- mice. Disorganized outer nuclear layer was observed in diabetic wt but not in diabetic Trpv4-/- retinas. We further demonstrate increased water diffusion, increased distances between photoreceptor nuclei, reduced nuclear area in all nuclear layers, and BRB hyperpermeability, in diabetic wt mice, effects that were absent in diabetic Trpv4-/- mice. Retinas of diabetic mice treated with PBS showed increased water diffusion that was not normalized by GSK2193874. ADC maps in nondiabetic Trpv4-/- mouse retinas showed restricted diffusion. Our data provide evidence that water diffusion is increased in diabetic mouse retinas and that TRPV4 function contributes to retinal hydro-mineral homeostasis and structure under control conditions, and to the development of BRB breakdown and increased water diffusion in the retina under diabetes conditions. A single intravitreous injection of TRPV4 antagonist is however not sufficient to revert these alterations in diabetic mouse retinas.

Dataset Information

Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina.

Publications

Adeno-Associated Viral Vector 2 and 9 Transduction Is Enhanced in Streptozotocin-Induced Diabetic Mouse Retina.

Similar Datasets

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