Retinitis Pigmentosa and Leber's Congenital Amaurosis

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Questions and Answers

What is a defining characteristic of pluripotent cells?

They can differentiate into any type of cell in the organism. (correct)

They are unable to self-renew.

They can only become blood cells.

They are found exclusively in adult organisms.

What method is used to confirm the pluripotency of embryonic stem cells?

Liver function tests

Teratoma assay (correct)

Immunological compatibility testing

Blood cell analysis

Which of the following statements is true regarding adult (somatic) stem cells?

They can divide indefinitely and self-renew.

They are found in the inner cell mass of the blastocyst.

They are multipotent and found in bone marrow. (correct)

They can differentiate into any cell type.

What is a major ethical concern associated with embryonic stem cells?

They involve the destruction of the embryo. Signup and view all the answers

Which of the following is true about the knockout mouse experiment?

It requires the alteration of genes in a blastocyst stage mouse. Signup and view all the answers

Which statement accurately describes the role of transgenes in gene therapy for dogs?

They comprise a gene and a promoter, usually a smaller sequence. Signup and view all the answers

What is a characteristic of stem cells that distinguishes them from differentiated cells?

They possess the ability to produce multiple types of cells. Signup and view all the answers

What type of cell division results in two cells of different types?

Asymmetric division. Signup and view all the answers

Which cells are considered totipotent in the context of cell potency?

Morulas. Signup and view all the answers

What determines the type of cell division and the identity of new cells?

Intrinsic and extrinsic signals. Signup and view all the answers

Which of the following is an example of a multipotent cell type?

Progenitor cells. Signup and view all the answers

In stem cell research, what does the term 'potency' refer to?

The capacity to produce a variety of cell types. Signup and view all the answers

What is the role of pluripotency genes in stem cells?

They indicate the ability to self-renew. Signup and view all the answers

What is the most common mutation associated with Retinitis Pigmentosa (RP)?

Mutation in rhodopsin Signup and view all the answers

Which of the following accurately describes Leber's Congenital Amaurosis (LCA)?

It can result from mutations in the RPE65 gene. Signup and view all the answers

What does the presence of increased number and size of vacuoles in RPE cells indicate in dogs with LCA?

Accumulation of all-trans retinal and nonfunctional RPE. Signup and view all the answers

Which genetic analysis technique is used to identify mutations in LCA?

Restriction digestion of PCR products Signup and view all the answers

What is required to successfully apply gene therapy for Leber's Congenital Amaurosis?

Alive non-dysfunctional cells. Signup and view all the answers

What role does the promoter play in gene therapy for inherited retinal diseases?

It directs transcription of the transgene. Signup and view all the answers

What is the consequence of losing photoreceptor cells in the retina?

Thinner retina. Signup and view all the answers

Which vector is commonly used for delivering gene therapy specifically to RPE cells?

Adeno-associated virus (AAV) Signup and view all the answers

What is the primary reason photoreceptor cell therapy for RP has not been successful?

Photoreceptors do not possess the ability to proliferate. Signup and view all the answers

What is the defining characteristic of induced pluripotent stem cells (iPSCs)?

They are produced by reprogramming differentiated adult cells. Signup and view all the answers

Which method is best suited for visualizing the presence of specific proteins in tissue sections?

Tissue immunostaining Signup and view all the answers

What crucial measurement can indicate the success of cell therapy in RPE replacement?

Retinal thickness Signup and view all the answers

Which method was pivotal in demonstrating that differentiated cells can be made pluripotent?

John Gordan's Somatic Cell Nuclear Transfer from frogs. Signup and view all the answers

Which of the following techniques helps confirm the expression levels of targeted cell proteins?

Percent protein expression levels Signup and view all the answers

Which of the following is NOT a potential application of iPSCs?

Cloning entire organisms. Signup and view all the answers

Why is it difficult to use somatic cell nuclear transfer for more complex animals?

There is low success with differentiation in complex animals. Signup and view all the answers

Why is it essential to ensure no other pluripotent cells are present during cell therapy evaluation?

Pluripotent cells may differentiate incorrectly. Signup and view all the answers

What does a Western Blot technique primarily detect?

Proteins Signup and view all the answers

Which cell type is not suitable for cell therapy due to the need for specific synaptic connections?

Neurons. Signup and view all the answers

What aspect of RPE cells is examined using electron microscopy in the context of therapy evaluation?

Polarization and structure Signup and view all the answers

What was the significant discovery related to pluripotency in iPSCs by Yamanaka?

Pluripotency genes can be activated using a virus. Signup and view all the answers

In which scenario would you likely encounter concerns about cell rejection?

When using hESCs Signup and view all the answers

What feature of iPSCs makes them similar to embryonic stem cells?

They can differentiate into any cell type. Signup and view all the answers

Which cells are mentioned as being able to be effectively replaced in cell therapy?

Retinal pigment epithelium (RPE) cells. Signup and view all the answers

Study Notes

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.

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Description

Explore the genetic underpinnings and characteristics of Retinitis Pigmentosa (RP) and Leber's Congenital Amaurosis (LCA). Delve into the common mutations, effects on vision, and notable differences in both humans and animal models. This quiz will deepen your understanding of these inherited retinal disorders.