1: intro to gene therapy

what is the transcriptome

collection of all RNA transcripts in a cell

what is the proteome

collection of all proteins in a cell or tissue

what are some gene therapy strategies

• replace missing gene product

• stimulate normal and/or block pathological processes

• eliminate or inactive toxic proteins

what does replacing a missing gene product entail

• adding back biosynthetic component like using a dietary supplement

• introducing a new gene into the affect cell

what does it mean to stimulate normal and or block pathological processes

• block proliferation of target cells if there is excess proliferation

• block blood vessel formation and growth for excessive neovascularization

• prevent cell death by adding survival factors or genes that block apoptosis

steps for replacing/adding a gene

1. identify mutation in patient

2. clone correct/normal gene

3. package DNA for normal gene in viral vector

4. virus infects cells and sends DNA to nucleus

5. virus DNA cargo expresses long-term and makes correct gene product

strategies for excess proliferation

• destroy neovascular tufts

• reduce blood vessel growth

• kill abnormal cells

• removal of tumor

• block proliferation

what is a way to destroy neovascular tufts

laser ablation

what is a way to reduce blood vessel growth

block vessel growth signaling pathways

what is a way to kill abnormal cells

chemotherapy

what is way to remove a tumor

surgery

what is a way to block proliferation

antibiotics

what are some solutions to excess cell death

• eliminate/inactivate toxic proteins

• add survival factors or genes to block apoptosis

what are some neovascular retinal disease

• wet AMD

• corneal neovascularization

• retinopathy of prematurity

• diabetic vacsulopathy

characteristics of neovascular ocular disease

• excessive blood vessel growth

• leakage and edema

• vision loss

• retinal detachment

therapies for neovascular ocular diseases

• eliminate abnormal vessels

• block blood vessel growth

• eliminate/disable signals for blood vessel growth

• manage insulin/sugar levels for diabetes

• hot laser destroys abnormal blood vessels

• con is that it results in blind spots

laser ablation

• systemic (venous) injection of verteporfin drug

• laser activates drug and forms blood clots to stop leakage

• usually used in combo with other drugs

photodynamic therapy

• inhibit, block, and/or destroy VEGFA

• cons: requires repeated intraocular injections and could block normal functions of VEGF

molecular therapy to block blood vessel growth

what leads to excessive production of VEGF

• hypoxia

• complement activation

• cellular damage/death

• disease

what does high levels of VEGF lead to

• vasoproliferation

• vasomigration

• vasopermeability

ultimately: CNV - choroidal neovascularization

what are aptamers

• first ocular anti-VEGF therapy

• short RNA oligonucleotides that bind to VEGF and inhibit function

• VEGF bound to aptamers cannot bind to receptors on endothelial cells

how are antibodies used in anti-VEGF therapy

antibodies bind to VEGFA so that VEGFs are not able to bind to receptors on endothelial cells

how are decoys used in anti-VEGF therapies

a soluble VEGF receptor protein binds excess VEGF to reduce the amount of free VEGF and prevent/reduce the activation of receptors on endothelial cells

what is RPE65

retinoid isomerohydrolase

an enzyme in the retinoid (visual) cycle that is required for processing retinal ester

what can mutations in RPE65 lead to

LCA - Leber congenital amaurosis

what is the chromophore in the retinoid cycle

• 11-cis-retinal

• when bound to opsin, it absorbs light and starts signal transduction in photoreceptor

• light isomerizes 11-cis-retinal to all-trans-retinal