Retinitis Pigmentosa and Leber's Congenital Amaurosis

Retinitis Pigmentosa (RP)

• Affects individuals of all ages
• Causes: mutations in at least 35 different genes
• Result: rod photoreceptor cell death
• Most common mutation: rhodopsin (20-30% of cases)

Leber’s Congenital Amaurosis (LCA)

• Affects people from birth or in the first year of life
• Rods and cones do not die
• Mutation in the RPE65 gene
• Fundus image appears normal
• Genes linked to LCA: CEP290, Guanylyl cyclase, RPE65

LCA in Briard Dogs:

• Increased number and size of vacuoles in RPE cells
• Vacuoles accumulate all-trans retinal due to nonfunctional RPE
• Leads to blindness
• Outer nuclear layer (rods) remain normal, no cell loss
• Normal fundus image
• Abnormal electroretinogram (ERG)
• DNA analysis shows a 4-base pair deletion, resulting in a frameshift mutation

LCA in Mice:

• Disrupted rod disk packaging, causing abnormal outer segments
• Rhodopsin remains in the disks, but reduced protein levels
• Rods do not die or disappear
• Normal fundus image
• Abnormal ERG
• Detected using immunoblot

Determining Disease Mutations

• DNA sequencing of wild type and mutant DNA
• Restriction digestion of a PCR amplified DNA fragment
• Pedigree analysis

RPE65

• Converts all-trans retinal back to 11-cis retinal.
• Mutations lead to significant vision issues

Arrestin

• Common protein that deactivates rhodopsin, a 7-transmembrane protein

Loss of photoreceptor cells

• Results in retinal thinning

Gene Therapy for LCA

• Gene therapy: used in humans
• Gene Rescue: used in animals

Necessary Components for Gene Therapy

• Wild Type Gene: delivers the functional gene
• Virus/Vector: transports the wild-type gene to the correct cells (RPE)
• Promoter: regulates gene expression, enabling transcription at the appropriate time
• Non-dysfunctional cells must be alive (can use for LCA, but not RP)
• Disease must be recessive, allowing for introduction of a dominant gene
• Injection location is crucial

Experimental Timeline:

• 2005: gene therapy trials conducted in Briard dogs at UPENN
• 2013: Spark Therapeutics identifies promoter and DNA for LCA treatment
• 2017: Clinical trial results published
• 2017: FDA approves Luxterna

Transgene

• Composed of gene (usually cDNA) and a promoter

Gene Therapy in Dogs

• No need for wild-type human RPE65 protein
• Requires a promoter, wild-type copy or cDNA, and a virus

Testing Gene Therapy:

• Trials on dogs or other animals
• Rigorous testing to ensure effectiveness before clinical trials
• Evaluation of various viruses and genes

Stem Cells:

• Undifferentiated: Not committed to a specific cell type
• Self-renewing: Divide and maintain undifferentiated state
• Potency: Capable of producing many types of cells
• Present throughout an organism's life

Types of Cell Division:

• Asymmetric: Produces two cells of different types
• Symmetric: Produces two cells of the same type
• Extrinsic (environmental) and intrinsic (intercellular) signals regulate division and cell identity

Potency in Different Cell Types:

• Morula: Totipotent, capable of becoming any cell in the organism and its extra-embryonic tissue
• Blastocyst (inner cell mass): Pluripotent, capable of becoming any cell in the organism
• Ectoderm/Mesoderm/Endoderm: Multipotent, capable of becoming any cell within their respective sublevels
• Progenitor Cells: Multipotent, commit to a range of similar cell types but cannot self-renew
• Precursor cells: Unipotent, can only commit to one specific cell type
• Differentiated cells: No potency, committed to their final, specialized cell type

Pluripotent Cells:

• Capable of developing into any cell type within an organism

Pluripotency Markers

• Oct4, Sox2, C-Myc, SSEA-4
• Expression levels assessed using gene expression analysis

Knockout Mouse Experiment:

• Embryonic stem cells containing an altered gene are injected into a blastocyst of a black mouse
• Resulting offspring possess the altered gene
• Breeding produces fully gene-manipulated mice

Determining Pluripotency:

• Teratoma Assay: Injection of pluripotent cells into a mouse or rat abdomen
• RNAseq or Microarray: Used to assess expression levels of pluripotency marker genes
• In Vitro Embryoid Bodies: Human ES cells cultured in vitro with growth factors, creating embryoid bodies

Adult (somatic) Stem Cells:

• Multipotent stem cells
• Found in bone marrow
• Present within the body
• Limited self-renewal capacity
• Lower risk of immune rejection

Embryonic Stem Cells:

• Pluripotent stem cells
• Found in the inner cell mass of the blastocyst
• Ethical concerns: destruction of the embryo
• Indefinite division potential
• Self-renewal capacity
• Potential for immune rejection
• Isolated from the blastula/blastocyst
• Capable of forming a teratoma when injected into a mouse
• Easier to culture

Commitment:

• A cell transitions to a lower potency state and cannot revert back
• Choosing to become a different cell type

Differentiation:

• Final commitment stage where the cell acquires its fully specialized form

Induced Pluripotent Stem Cells (iPSCs):

• Pluripotent cells generated by treating differentiated adult cells with specific factors
• Induced to re-express pluripotency genes
• Similar to embryonic cells, but retain cellular memory
• Applications:
  • Studying cell development
  • Investigating disease progression
  • Cell Therapy
  • Drug Development

History:

• John Gurdon's Somatic Cell Nuclear Transfer (Frog Cloning):
  • Nuclear transplantation from a frog's epithelial cell to an enucleated egg cell
  • Led to the development of iPSCs

Yamanaka and iPSCs:

• Took mouse fibroblast cells and introduced a transgene containing pluripotency genes
• The transgene promoter activated when cells became pluripotent
• Discovered:
  • Pluripotency alters cell morphology and gene expression

Cell Therapy:

• Replacement of old or dead cells with new ones (cannot be used for neurons)
• Successfully used for RPE cells

Cell Therapy for AMD:

• Uses hESCs, iPSCs, or RPE cells to produce RPE cells that are then injected into the eye

Cell Therapy for RP:

• Unsuccessful due to the neuronal nature of photoreceptors

Determining Success of Cell Therapy:

• Immunostaining: Detects expression of targeted cell proteins
• Protein expression levels: Ensures consistent expression
• Electron microscopy: Verifies cell morphology and structure
• Functional tests: Evaluates cell function (e.g., RPE phagocytosis)
• Absence of other contaminating pluripotent cells
• Examination of retinal thickness: Increased thickness indicates a new RPE layer

BCVA

• Tests confirm vision and reading improvements

Cell Therapy Concerns:

• Potential rejection, especially from hESCs

In vitro:

• Culture in a dish

In vivo:

• Animal studies

De Cruz Study (fixing mouse retina):

• Demonstrated:
  • Production of stable RPE cells
  • Absence of pluripotent or multipotent cells
  • Non-proliferative nature of RPE cells generated from stem cells

Interpreting Data from Figures:

Western Blot (Immunoblot)

• Used to detect and quantify a specific protein within a biological sample
• Band size and intensity indicate protein expression levels

Polymerase Chain Reaction (PCR)

• Amplifies a specific DNA sequence for analysis
• Identifies the presence or absence of certain DNA fragments or evaluates size changes
• Helps detect missing codons and mutations

Reverse Transcriptase PCR (RT-PCR)

• Detects and quantifies RNA by converting it into cDNA, facilitating gene expression analysis

Tissue Immunostaining

• Visualizes specific proteins or cells within tissue sections

• Provides information about cell location and structure

• Utilizes antibodies to stain thin sections of retina and highlight microglia

• Note: The figures have not been provided; therefore, the last bullet item is incomplete.