Gene therapy involves introducing genes into cells to treat disease. CRISPR is a gene editing technique using Cas9 protein and guide RNA. It allows DNA cutting and modification. Luxturna is a gene therapy for an inherited eye disease caused by RPE65 gene mutations. It uses AAV vector to insert a healthy RPE65 gene into retinal cells. In clinical trials, Luxturna improved patients' visual function and mobility in low light after 1 year as measured by a mobility test.

1. Gene Therapy
Ruqaya Anani

2. Contents
Gene Therapy01
Genome Editing02
CRISPER03
Luxturna04

3. Gene Therapy
Gene therapy can be defined
as the genetic modification of
DNA to produce a therapeutic
effect by replacing a mutated
gene, adding a missing gene
or modifying an existing one.

4. Gene Therapy
It involves the introduction
of one or more foreign
genes into an organism to
treat hereditary or acquired
genetic defects.

5. Why is Gene Therapy important
Gene therapy can be used for a
number of diseases, such as
severe combined immune-
deficiencies, hemophilia,
Parkinson's disease, cancer and
even HIV through a number of
different approaches.

6. Types of gene
therapy
01
02SOMATIC CELL
THERAPY
GERM LINE
THERAPY

7. SOMATIC CELL GENE
THERAPY
Therapeutic genes transferred
into the somatic cells.
Will not be inherited later
generations.
At present all researches
directed to correct genetic
defects in somatic cells.
Ex. Introduction of genes into
bone marrow cells, blood cells,
skin cells etc.
A
B
C
D

8. GERM LINE GENE
THERAPY
Therapeutic genes transferred
into the germ cells.
It is heritable and passed on to
later generations.
For safety, ethical and technical
reasons, it is not being
attempted at present.
Ex. Genes introduced into eggs
and sperms.
A
B
C
D

9. Two ways to deliver genes
In vivo approach
Ex vivo approach

10. EX vivo VS. In vivo
EX vivo
Cells are modified outside the
body and transplanted back in
again.
This approach can be applied
to the tissues like
hematopoietic cells and skin
cells which can be removed
from the body.
In vivo
Genes are changed in cells
when the cells are in the body.
This is done in case of tissues
whose individual cells cannot
be cultured in vitro in sufficient
numbers (like brain cells)
and/or where re-implantation of
the cultured cells in the patient
is not efficient.

11. Ex Vivo

12. In Vivo

13. COMMON VECTORS USED FOR GENE THERAPY
Retro viruses Adeno viruses Liposomes

14. Genetic Improvement and modification
01
02
03
04
05
Conventional Breeding
Mutational Breeding
Transgenic
Epigenetic Modifications
Genome Editing

15. Genome editing or genome editing with engineered
nucleases (GEEN)
It is the insertion, deletion or replacement of
DNA at a specific site in the genome of an
organism or cell.
It is usually achieved in the lab using
engineered nucleases also known
as molecular scissors

16. 02
01
03
Genome editing or genome editing with engineered
nucleases (GEEN)
Zinc finger nucleases (ZFNs)
TAL effector nucleases
(TALENs)
CRISPRs
Methods:

17. A palindromic sequence is a nucleic acid sequence
(DNA or RNA) that is the same whether read
5' (five-prime) to 3' (three prime).
Repeats: are patterns of nucleic acids (DNA or RNA)
that occur in multiple copies throughout the genome.
CRISPRs
(Clustered Regularly Interspaced Short
Palindromic Repeats)

18. DNA loci containing short repetitions of base sequences.
Each repetition is followed by short segments of "spacer DNA" from
previous exposures to a virus.
CRISPRs are found in approximately 40% of
sequenced bacteria genomes and 90% of sequenced archaea.
CRISPRs

19. CRISPRs/Cas
CRISPRs are often associated with cas genes that code for proteins
related to CRISPRs.
The CRISPR/Cas system is a prokaryotic immune system that confers
resistance to foreign genetic elements such as plasmids and phages
By delivering the Cas9 protein and appropriate guide RNAs into a cell,
the organism's genome can be cut at any desired location.

20. CRISPR/Cas system who it works?

21. CRISPR/Cas system who it works?

22. CRISPR/Cas system who it works?
A copy of the invading nucleic acid is integrated into the CRISPR locus.
CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus.
crRNAs are then incorporated into effector complexes, where the crRNA
guides the complex to the invading nucleic acid and the Cas proteins
degrade this nucleic acid.

24. Application of CRISPR
By blocking RNA polymerase at the promoter region of the DNA.
CRISPRi has the ability to silence multiple genes at the same time
CRISPR Interference (CRISPRi)
Single Gene Knockout System
Multiple Gene Knockout Model
Organisms
Gene Knockout System
Many genetic diseases could be cured
By modifying genomic sequences of
pluripotent stem cells of patients to express
wild-type copies of the disease causing
genes
Gene Knockin System

25. Application of CRISPR

26. CRISPR/Cas9 is a novel technique with a bright future
in genomic editing.
CRISPR is still a young system and more research
must be completed in order to rectify its problems.
It has the potential to be useful in a broad range of
applications from simplifying research to acting as a
new form of gene therapy for patients with HIV and
genetic diseases.
Research in the area of CRISPR/Cas9 is gaining
speed and this system could very well be the
solution to many medical issues we face today
CRISPR

28. Introduction
LUXTURNATM (voretigene neparvovec-rzyl)
is a prescription gene therapy
product used for treatment of
patients with inherited retinal
disease due to mutations in both
copies of the RPE65 gene.

29. LUXTURNATM
RPE65 is a gene that is necessary to
make a protein needed for vision.
RPE65 gene mutations cause vision
to deteriorate and may progress to
complete blindness.

30. LUXTURNATM
RPE65 is 1 out of more than 260
genes that maybe responsible for an
inherited retinal disease.
Confirming a gene mutation with a
genetic test is the first step to
learning if LUXTURNA may be
right for you.

31. Night blindness (nyctalopia)
Loss of light
sensitivity
Loss of sharpness or
clarity of vision
Impaired dark
adaptation
Repetitive uncontrolled
movements of the
eye (nystagmus)
If you have mutations in both copies of the RPE65 gene,
you may experience:

32. Mechanism of action
LUXTURNA introduces a functional copy of the RPE65 gene to compensate for the
RPE65 mutation.
1 2
3
RPE65 gene delivery. RPE65 protein production.
Restoring the visual cycle.

33. Adeno-Associated Virus

34. Mechanism of action
First step:
LUXTURNA uses the
adeno-associated viral
Vector serotype 2 (AAV2)
to carry a functional
Copy of the RPE65 gene
into the retinal pigment
Epithelial (RPE) cells to
compensate for the
RPE65 mutation.
RPE65 gene delivery

35. Mechanism of action
Second step:
With a functioning
RPE65 gene, the
cells begin
producing the
RPE65 protein.
RPE65 protein
Production:

36. Mechanism of action
Third step:
With the functional
RPE65 protein,
11-cis-retinal
regenerates to
restore the visual
Cycle.
Restoring the visual
cycle:

37. How is LUXTURNA administered?
LUXTURNA is given by a healthcare
professional as a surgical injection
beneath the retina of each eye.
One eye is treated at a time. After the
first eye is treated, the second eye will
be treated at least 6 days later.

38. How was the effect of LUXTURNA
measured?
Visual Acuity
Visual Field
Light Sensitivity
You may be used to tests such as:
These tests measure visual function, which is how vision loss affects your eyes.

39. Multi–Luminance Mobility Test
A new test was created to
measure functional vision,
which is how vision loss affects
the ability to perform daily life
activities.
This test is called the Multi-
Luminance Mobility Test.

40. Multi–Luminance Mobility Test
In the Multi-Luminance Mobility
Test, participants had to
navigate a course of obstacles
under different light levels.
The light levels went from
bright to dark.

41. Multi–Luminance Mobility Test
• LUXTURNA was administered to
the participants' first eye, and 6-
18 days later, LUXTURNA was
administered to their second eye.
• Participants followed a
prescribed course of medication
to reduce risks related to an
immune response.

42. Multi–Luminance Mobility Test
• Each participant was evaluated 1
year after treatment to measure
the effect of LUXTURNA
• LUXTURNA has 9 years of safety
experience and has been studied
in a range of patients between 4
and 44 years old.

43. What were the results?
LUXTURNA improved functional vision. This
increased participants' ability to perform activities
of daily living.
One year after treatment, participants were able to
navigate through the Multi-Luminance Mobility Test
course using both eyes at 2 light levels
darker than before treatment.

44. What were the results?
A majority of participants
were able to complete the
course at the darkest light
level after treatment.

46. Thank you
Any Question ?