Measuring adeno-associated virus improvements with Phoenix MICRON™ fluorescent imaging and Phoenix MICRON™ Ganzfeld ERG

A team of researchers at the Indian Institutes of Technology have published three detailed articles examining how to improve adeno-associated viruses (AAV). Maurya, S, Mary, B, Jayandharan, GR et al -approach the improvement of the viruses in a stunningly detailed gene-to-cell-to-whole-mouse model, narrowing down a multitude of options and producing impressive fluorescent fundus images and Ganzfeld ERG traces with the Phoenix MICRON and Phoenix Ganzfeld ERG.

fundus imaging of murine eyes
Figure 1. Ocular gene transfer in C57BL6/J mice with SUMOylation site–modified K105Q vector. A fundus imaging of murine eyes was performed 4 weeks after administration of AAV2wild-type and AAV2 K105Q vectors. For these experiments, we used a Micron IV imaging system (Phoenix Research Laboratories, Pleasanton, CA) that employs a standard mouse objective and a field of view at 50_ (1.8mmdiameter). Intensity was set at maximum and gain was set at 15 db; the frame rate was set at 6 fps for imaging of all the groups. Image analysis was performed by using Concentric Circle Plugin in the ImageJ software, and AAV2 K105Q group had a 1.57-fold higher EGFP expression in comparison to AAV2 wild-type eyes (n = 4 to 7 eyes). In “Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis.”

Adeno-associated viruses are essential therapeutic tools that have already shown promising results in improving genetic conditions such as hemophilia and Leber’s congenital amaurosis. However, since they are viruses, they can set off negative immune responses which results in treatment at sub-optimal levels. The team of researchers sought to improve the efficacy of adeno-associated viruses paired with green fluorescent protein (GFP) and/or RPE65. GFP allowed measurement of the increase in fluorescent protein expression with Phoenix MICRON fluorescent imaging. RPE65 treated visually impaired mice which whose vision rescue was measured with the Phoenix Ganzfeld ERG.

In “Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis,” Maurya, S, Mary, B, and Jayandharan, GR increased the virus efficacy by altering ubiquitin-like modifiers. When linked with GFP, this improved virus showed a 1.6-fold increase in GFP expression as examined with the Phoenix MICRON fluorescent imaging (Fig 1). When the improved virus was paired with RPE65 and injected sub-retinally into a mouse model for Leber’s congenital amaurosis (rd12 mice), a genetic disorder resulting in severe vision loss or blindness, the mice demonstrated improved visual function as measured by Ganzfeld ERG compared to mice injected with the original virus paired with RPE65 (Fig 2).

In “Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration,” Mary, B, Maurya, S et al altered glycosylation sites to improve gene expression. Phoenix MICRON fluorescent images revealed a 2-4 fold increase in GFP in mice injected with the improved virus paired with GFP (Fig 3). The Phoenix Ganzfeld ERG revealed a higher rate of photoreceptor response in a mouse model of Leber’s congenital amaurosis injected with the improved virus paired with RPE65 than those injected with the original virus (Fig 4).

Adeno-associated viruses go through post-translational modifications that are poorly understood and may affect the efficacy of the vector. In “Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes,” Mary, B, Maurya, S et al examined several different types of post-translational modifications and test the effects. One post-translational modification lead to lower expression of GFP as seen with the Phoenix MICRON (Fig 5).

Article citations:

Maurya, S, Mary, B, Jayandharan, GR. (2019). Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis. Hum Gene Ther. 30(12):1461-1476.

 

Mary, B, Maurya, S, Kumar, M, Bammidi, S, Kumar, V, Jayandharan, GR. (2019). Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration. Mol Pharm. 16(11):4738-4750.

 

Mary, B, Maurya, S, Arumugam, S, Kumar V, Jayandharan, GR. (2019). Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes. FEBS J. 286(24):4964-4981.

ERG Recordings
Figure 2. SUMOylation site–modified vectors expressing RPE65 demonstrate phenotypic rescue in rd12 mice model. Eyes of rd12 mice were mock-injected or injected with ssAAV2-RPE65 and ssAAV2K105Q-RPE65 vectors at a dose of 7 · 108 vectors via subretinal route. Scotopic ERG recordings were performed 10 weeks postvector administration and their representative image (a) and quantification data (b) are shown. Completely opaque eyes caused by injury were eliminated from the recording data set. Dunnett’s multiple comparisons test was used to determine the statistical significance. Data are expressed as mean – SD (n = 11–13 eyes per experimental group). ***p £ 0.001. In “Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis.”
gene expression measured by fundus imaging in a Micron IV imaging system
Figure 3. Ocular gene transfer efficiency of mutant AAV2 vectors. About 3 × 108 vgs of either scAAV2-WT or AAV2 mutants were administered intravitreally into the eyes (n = 6) of C57BL6/J mice. Four and six weeks after ocular gene transfer, the gene expression was measured by fundus imaging in a Micron IV imaging system. Representative data of (A) 4 weeks after ocular gene transfer from scAAV2-EGFP, scAAV2-T14N, scAAV2-Q259N, and scAAV2-N705Q vector injected eyes are shown. The quantitative data of (A) are represented in (B), after image J analysis (n = 4). The fold difference in mean GFP intensity is provided in comparison to scAAV2-WT at 4 and 6 weeks. **p < 0.01 vs WT-AAV2-injected
mice. In “Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration.”
ERG Wave Forms
Figure 4. Visual function rescue in rd12 mice by AAV2 T14N mutant vector. (A) Representative images of the rescue in ERG wave forms in AAV2-T14N-injected eyes when compared to wild type vector injected and mock controls in rd12 mice after 6 weeks and (B) 10 weeks. A rescue in physiological vision as represented by the regain in qualitative wave form was noted. (C) Dot plot for “a wave” and “b wave” plotted against the mean amplitude obtained at 3.1 log cd s/m2 shows significant rescue in “a wave” form (left graph) and “b wave” (right graph) in the mutant vector injected group when compared to wild type vector injected and mock controls at 6 weeks and (D) 10 weeks post gene transfer. n = 4−6 eyes. Values represented are mean ± standard deviation. **p < 0.01, ***p < 0.001. In “Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration.”

 

fundus imaging of GFP expression
Figure 5. Ocular gene transfer efficiency of AAV2-WT and AAV2-K258Q vectors was assessed by fundus
imaging of murine eyes, 2 weeks after intravitreal gene transfer with vectors. The standard fundus imaging in the Micron IV system (Phoenix Research Laboratories, Pleasanton, CA, USA) was performed with a mouse objective and a field of view at 50° (1.8-mm diameter). The images demonstrate GFP expression from representative eyes (n = 4) from a total of six eyes administered for each group. All the
images were captured at a uniform exposure setting for both the groups [Intensity at maximum, gain at 18 db (decimal gain), frame rate at 2 fps (frames per second)]. In “Post‐translational modifications in capsid proteins of recombinant adenoassociated virus (AAV) 1‐rh10 serotypes.”