AMANDA GUARISCO

INHERITED BLINDNESS

LEBER’S CONGENTIAL AMAUROSIS

• A rare inherited eye disease; affecting 1 in 80,000• Due to mutations of several genes affecting the

development of the retina of the eye, most importantly the RPE65 gene• Little Jenny Donohue, a bright, optimistic seven

year old girl suffers from LCA• Her parents noticed she had poor vision and

nystagmus eye movements in her early years, but as she got older, her vision got progressively worse

NORMAL VS. LCA RETINA

JENNY’S STORY

• Ever since she was little, she loved watching basketball with her father and wanted to play on her school team when she got old enough. Neither she nor her family expected her to have this disease; they were unprepared for a lifetime of hardships and limitations this would cause.• Her ignorant parents did not even know LCA

existed, even though they were both carriers. Therefore, they were never tested for any abnormalities in their DNA. Now, they decide to have Jenny’s DNA tested because they heard there was a chance she could be cured and fulfill her dream of playing on the team.

• Luckily, Jenny lived in Iowa and at the University of Iowa hospital, they have DNA testing available. The lab draws Jenny’s blood and begins the basic testing with Southern blotting which is a method routinely used for detection of a specific sequence in DNA samples. It uses gel electrophoresis-separated DNA fragments to identify the specific genes causing LCA using DNA probes.

• Further testing was then performed using the microarray chip mutation detection that identifies 32% of LCA sequence variants

• Research into the cause of LCA has been helpful in treating the disease.• DNA Sequence Analysis is clinically available for

11 of the 12 of the genes associated with LCA, including the RPE65 gene.• Deletion/Duplication Analysis is also clinically

available for the RPE65 gene. Research has shown that Partial and Whole gene Deletion/Duplication has been identified as the mutation in this gene.

• 3 -16% of LCA is due to a mutation on the gene RPE65 although 12 other genes have been identified as a cause• Gene therapy is currently the only cure for the

disease and has proven effective in clinical trials• Gene therapy essentially replaces the abnormal

gene with a normal one

• The Medical Center identified her mutated RPE65 gene as the cause of her illness. • Jenny had the option of undergoing gene transfer

surgery to help fix her vision using rAAV(recombinant adeno-associated virus) as a vector to deliver the normal DNA. • The idea was to target the vitreous fluid in her

eyes in order to inject millions of copies of healthy RPE65 genes so they could get to the retinal cells.• Following the delivery of rAAV, the vectors

effectively transfer normal DNA into RPE(retinal pigmented epithelium) and photoreceptor cells.

VECTORS

• A vector is any agent that acts as a carrier or transporter, such as a virus or plasmid that conveys a genetically engineered DNA segment into a host cell.

• A normal RPE65-carrying virus vector, known as the adeno-associated virus (AAV), was engineered as a safe vector to carry the new, healthy gene to its target.

• In Jenny’s case, the target was the cells of the retina. Following the delivery of rAAV, the vectors effectively transfer normal DNA into RPE(retinal pigmented epithelium) and photoreceptor cells.

COPYING DNA

• To create these normal gene copies, a process known as polymerase chain reaction (PCR) was used to amplify DNA in vitro by incubating it with special primers, nucleotides, and DNA polymerase molecules.

• mRNA and its cDNA(complementary DNA) for the normal RPE65 gene were used as templates in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase to make the normal copies.

• This process makes billions of a target segment of DNA in just a few hours which will then be incorporated into the vector.

• The University research scientists and physicians created the normal RPE65 gene copies and the vectors needed to deliver them to the target cells. • Jenny was lucky enough to have the procedure

done. The vitreous humor of Jenny’s eyes were injected with the treatments.

RESULT

• Throughout the post procedure years, she has shown improvement in both of her eyes. She had regained little vision back, but doctors say she will never regain full eyesight for the rest of her life.• Unfortunately, she will never see well enough to

play basketball for her school, but her attitude never gets down and she considers herself lucky to now not be completely blind

SOURCES

• http://www.ncbi.nlm.nih.gov/books/NBK1298/#lca.Therapies_Under_Investigation• http://www.nature.com/gt/journal/v11/n1s/full/

3302366a.html• http://www.ncbi.nlm.nih.gov/pubmed/18441371• http://www.nei.nih.gov/lca/