Exam assay Flashcards
What is the title?
Proposing older RPE65 Briard dogs as a promising animal model when testing sub-retinal rAAV2 gene therapy in Leber Congenital Amaurosis.
What is the hypothesis?
We propose that injecting an rAAV2 hybrid vector containing RPE65 gene into the sub-retina will provide electroretinogram and vision testing performance improvements in the test eye of the older Briard dog model, when compared to the contralateral control.
How common is LCA?
—very rare autosomal recessive condition
—estimated to affect 1 in 40,000 Australians
—5% of inherited retinal dystrophies
How would you explain the LCA condition?
—characterised by progression vision lost
—onset in early infancy
—caused by a dysfunction in the RPE65 gene which distrupt retinal cycle
—rod and cone photo-receptors degenerate and normal vision pigmentation cannot occur leading to significant vision loss
—severe cases patients can have little or no light perception at all
Is there a cure for LCA?
No effective treatment for this disease
What previous research has investigated this issue?
—Previous research has investigated using gene therapy to deliver a functional copy of the gene to the retina.
—in early trials of sub-retinal therapy in a naturally occurring RPE65 deficient dog model (Briard dog species)
—the results showed a dramatic improvement in photoreceptor function and vision testing performance
Why was this previous issue not successful?
—results were not replicated in human trials
—Briard dogs model experiences photoreceptor degeneration at a slower rate to humans
—previous research only investigated young Briard dogs (aged 2 years old)
—using a younger model of Briard dog does not provide a comparable animal model of the disease
What do we propose to do for our experiment?
—aim to investigate the success of delivery RPE65 gene in older Briard dog model (5-8 years old) on restoring vision
—we will be administering RPE65 gene via a rAAV2 hybrid vector with the assistance of an adeno-associated type 5 helper virus into the sub-retina of the left eye and the right contralateral eye will function as the measurable control.
—this will provide valuable information on the “window of opportunity” for treatment, as well as a more accurate picture of human dosage requirements
What will you include in the Animal section of methods?
—12x Briard dog chosen for study,
—3x 5 yr old, 3x 6 yr old, 3x 7 yr old, 3x 8yr old
—left eye testing, right eye contralateral control
—dogs will be treated with topical, subconjunctival and oral anti-inflammatory medication administered both preoperatively and postoperatively
—ethical guidelines and approval in accordance with the Animal Ethics Committee at the UWA.
—animals will be treated in accordance with the Australian code for care and use of animals for scientific purposes.
How will you construct the viral vector?
—A recombinant rAAV2 vector with adeno-associated type 5 helper virus
— human RPE65 cDNA coding sequence
—driven by the human RPE65 promoter
—flanked by AAV2 ITRs encapsulated in an AAV2 shell.
—B50 packaging cell line
—AAV requires a helper virus for replication, generally considered non-pathogenic and once cell is transduced, it remains episomal, reducing the potential for genomic integrating events.
—AAV can infect both non dividing and dividing cells with persistent expression, making this an attractive vector to use.
—The rAAV2 titers will be determined by dot blot assay generating concentrations of 1x10^12 vg ml^-1.
What is involved with the dot blot assay?
—Following production, culture media and cell lysates are subjected to dot blot to quantify the viral genome within the capsid shell.
—The first step of the dot blot assay is to treat the samples with a nuclease to remove contaminating plasmid DNAs and unpackaged AAV genomes in samples.
❏ Failure to do this first step would increase the
background signals in particular when unpurified
samples are assayed.
—This is then followed by a protease treatment to break viral capsids and release nuclease-resistant viral genomes into sample solutions.
—Next, viral genomes are denatured, blotted on a membrane, and hybridized with a viral genome-specific DNA probe for quantification.
Injections
What area, why did you choose it, concentration amount..?
—Injections are to be conducted in the central and dorsal regions of tapetal fundus of the intraocular reflective tissue.
—This site was chosen as the central retina in the normal canine has a greater number of photoreceptors per unit area, particularly of cones, than the peripheral retina, making it analogous to the human macula.
—In RPE65-deficient dogs in particular there is relative preservation of the outer nuclear layer in the central and superior retina with a more severe degeneration peripherally.
— This region enhances scotopic vision by reflecting light that has passed through the retina to stimulate photoreceptors.
—Injection will be administered at full concentration of 1 x 10^12 vg ml^-1.
Ophthalmic investigation and fundus imaging
—Ophthalmology examinations including slit-lamp biomicroscopy with aqueous humour flare testing, indirect ophthalmoscopy and wide field fundus testing.
—Examinations will be performed preoperatively, immediately postoperatively to test for ocular inflammatory responses as well as testing postoperatively and 4 weekly thereafter.
—Imaging for fundic abnormalities and tapetal reflectivity of subretinal injection sites.
Electroretinography
To assess cone and rod photo-receptor functional improvement, ERG will be performed preoperatively and 4 months postoperatively.
❏The preoperative ERG will be used to establish the flash intensity threshold for a response in A and B waves.
❏ Post-operatively a gradually increasing flash intensity beginning lower than the pre-treatment threshold will be used.
❏The first intensity that elicits an ERG B waveform will be noted to compare ROD photoreceptor improvement
❏The first intensity to elicit an ERG A waveform will be noted to compare CONE photoreceptor improvement.
Vision testing
To evaluate improvements in vision the contralateral eye will be covered with a blindfold and dogs will undergo a four-choice vision test.
❏ Each animal will enter a light-proof box with four exit tunnels, one of which is randomly selected to be open.
❏ Light will be delivered at varying intensities (full room light, medium and dim)
❏ The number of correct exit choices and average time to exit (7 attempts at each light intensity) will be recorded.
❏ This process will be performed pre- and post-operatively and in both the test and control eye.
