gene therapy the past the present and the future

1. Introducedto you
GENE THERAPY
By Mahmoud saad

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Slides contents
Limitations and Challenges .
Gene Therapy for Several Diseases
Process
Requirements
1. Gene delivery systems
2. Gene Editing
Introduction
1. Definition
2. Types of Gene
Therapy

3. GENE THERAPY
INTRODUCTION
WHAT IS GENE
THERAPY?
 Gene therapy is the science of making specific changes in
human genome in order to improve it or reach
therapeutic effects in gene-related diseases.
 These changes include replacing and editing
mutated genes or even introducing a normal
copy of genes to cells to bring back the normal
function of proteins
03

4. Gene therapy
is categorized into
three major groups
A
Germ line gene therapy
C
Vaccination gene therapy
B
Somatic gene therapy
Types of gene therapy
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5. 01
02
03
04
Safety concerns which can lead to off‐target
mutations.
Gene therapy has the potential to change the germ line
which has negative effects for future generations.
Misusing for human enhancement and eugenic purposes
which insert genes that would not occur naturally.
Changing the germ line and affect the next
generation without their consent
A. Germ line gene therapy
Genes are introduced into
sperm or egg cells and is not
widely applied because of
ethical issues such as:

6. 01
02
03
Somatic gene therapy has two primary
techniques: in vivo and ex vivo
The manipulated genes do not get passed on to the
other generations.
It Takes place at only identified body cells.
A. Somatic gene therapy
The transmission of a
gene to a body cell that
does not facilitate
reproduction so,

7.  Ex vivo gene therapy is based on collecting cells
from a donor, transducing cells with target gene and
finally introducing cells to patient body
 In vivo gene therapy is summed up in direct
introduction of functional and normal genes to
patient cells by different types of vectors.
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Somatic gene therapy

8. Three steps of gene therapy process
Step 01
The first phase of
doing gene
therapy is to
evaluate the
cause of an
infection.
Step 02
involves creating a
duplicate DNA
which matches the
defect gene to solve
the health issue.
Step 03
create a system
that can deliver
the new gene
into a patient’s
human body.
Find defect Gene Editing Delivery

9. GENE EDITING
One of the core steps of gene therapy is gene editing
and gene silencing; step in which scientists make
intended changes on human genome in order to
 study genes functions
 induction of missed functions
 repairing mutated gene causing a disease.
There are different techniques of gene editing
with different strategies which use different gene
editing tools with different purposes.
GENE KNOCK OUT
GENE KNOCK
DOWN

10. GENE KNOCK DOWN
 Reduction of the expression of one or more
genes of an organism but no completely
eliminated. This is done by degrading or
blocking translation of the gene’s mRNA
transcript.
 Some mRNA may remain intact and translated
so there is still low level gene expression.
 RNAi (RNA interference) can be used as gene
editing tool for gene knockdowns.
RNAi (RNA interference)
 Group of mechanism that uses
small RNA molecules to direct
gene silencing.
 Many organisms use RNAi to control
genes expression.
 Eukaryotic cells have many
sophisticated ways of controlling
gene expression in the very complex
environment of the cell these
mechanisms need to be precisely
targeted.

11. RNAi
mechanism
 There are several types of regulatory
small RNA, small interfering RNAs
known as siRNA are derived from
longer double stranded RNAs that
are either produced in the cell itself
or in gene editing are delivered into
cells experimentally.
 miRNAs are another type of small
RNA most miRNA come from RNAs
that are transcribed in the nucleus.
which then fold and processed
before being exported into the
cytoplasm as double stranded
precursor RNA.

12. After transcription of
target gene inside the
nucleus the transcript
mRNA is exported to
cytoplasm
01

13. The double stranded
precursors of miRNA and
siRNA bind to Dicer an
endonuclease protein
that cuts the RNA into
short segments.
02

14.  The short ds miRNA and
siRNA then bind an
Argonaut protein.
 one strand of the RNA is
selected and remain bound
to Argonaut this is called
the guide strand
03

15. the combination of
the RNA an Argonaut
along with other
protein is called the
RNA induced
silencing complex or
(RISC).
04

16. siRNAs direct RISC to bind to
specific mRNA of interest this
targeting is precise because it
is determined by base pairing
between the siRNA and the
target mRNA.
05

17. siRNAs often have perfect
complementarity to their
target sites. once bound
Arganaut catalyzes cleavage
of the mRNA which then be
degraded.
06

18. miRNA also guide RISC to
the target mRNA with
imprecise matching allows
miRNAs to either degrade
or inhibit translation of
mRNA leading to transient
silencing.
07

19. A
E L
Experimental
Validation
Applications Limitations
RNA Interference

20. Experimental Validation
T
O
W
E
Experimental
Validation
 In term of experimental validation in RNAi
system gene knockdown can be assisted by
measuring mRNA levels using qRT-PCR and
protein levels using Western Blot.
 If mRNA levels are decreased but protein
levels remain the same as before experiment,
protein turnover may simply be slow.
 If mRNA levels remain the same but protein
levels decrease, this means that the siRNA
may be inhibiting translation rather than
degrading mRNA

21. RNAi Applications
T
O
A
Applications
1. RNAi most important application is screening
of mammalian gene function
2. it could be used for treating viral infections
and cancer.
3. Giving that virus uses human cell machinery to
replicate RNAi may be used to generate viral-
resistant host cells by attacking viral and
human genes which are necessary for viral
replication.
4. Oncogenes, which accelerate cancer growth,
can be targeted by RNAi. This technique can
be used to prevent tumor growth by targeting
molecules important for neovascularization

22. LIMITATIONS
T
L  RNAi can cause significant Off-targeting effects,
one siRNA can potentially repress hundreds of off-
target mRNA transcripts as it does not require strict
sequence complementarity to bind.
 Unmodified siRNA are easily degraded by RNases
 Assays usually are of short duration (transient
inhibition only)
 Transcripts with high turnover are sometimes
difficult to silence
 Cost (especially at genome-wide level
Although RNAi very useful gene
knockdown tool it has its own limitations
as;
LIMITATIONS

23. GENE KNOCK OUT
 Gene knockout (KO) is a genetic technique in which
one of an organism's genes is made inoperative or
when a gene has acquired a frame shift mutation
such that the cell no longer expresses any functional
protein.
 When a double stranded break in the DNA is created, the
cell repairs it via NHEJ pathway. This process creates
insertions or deletions that can cause a frame-shift
mutation that eliminates gene expression.
 Knockout organisms are used to study gene
function, usually by investigating the effect of gene
loss.
CRISPR/CAS9
the most common and more precise
recently discovered gene editing tools that
have made a revolution in the field of gene
manipulation and allowed scientists to
perform conditional gene knock out to
eliminate a specific gene in a certain tissue.

24. Mechanism
Nickase Cas9
Cas9 Double
Mutant
saCas9
CRISPR/Cas9
A
B
C
D

25. Cpf1 Nuclease
Advantages
Limitations
Applications
CRISPR/Cas9
E
F
H
G

26. Massive
X
presentation
to
DesignBall
team
Massive
X
26
b
C
c
CRISPR/Cas9
 The CRISPR Cas9 system was originally observed as
bacterial adaptive immune system that provides
acquired immunity against foreign viruses and plasmids.
 Upon invading bacterial cells, viral genetic material
could be integrated into a CRISPR position to be
transcribed into CRISPR RNA (crRNA) which eventually
makes a complex with trans-activating crRNA and Cas9
endonuclease.
 This complex cuts foreign DNA into ds-DNA with blunt
end specifically at intended sequence incorporated
within crRNA.
 To function CRISPR/CAS9 must recognize crRNA
sequence and another sequence called protospacer-
related motif sequence or PAM.

27. MECHANISM
Step01

28. Step02
MECHANISM

29. MECHANISM
Then the cell uses its own repair mechanism
to repair the double strand break created by
Cas9 through :
Step03
a. Non-homologous end joining DNA
(NHEJ) repair pathway
 in the absence of a repair template.
 the ends of the DNA are simply
ligated back together
 leading to the introduction of small
insertion or deletion mutations
 disrupting the reading frame of the
desired gene.
b. Homology directed repair (HDR)
pathway
 In the presence of a repair template.
 This template will have homology to
the flanking regions of the double
strand break.
 This method of repair is highly
accurate and could be used to
introduce specific nucleotide
changes into the targeted gene.

30. MECHANISM

31. MECHANISM

32. MECHANISM

33. Cas9 Double
Mutant
Cpf1
Nuclease
saCas9
Nuclease
Different types of
Cas9 Nuclease
Cas9 Nickase

34.  This type was made by mutant form Cas9
protein in which one of the two endonuclease
domains was inactivated leading two
introduction of single strand nick instead of
double strand cleavage.
 developed to overcome off-target effects which is one of
the biggest concerns regarding gene editing using CRISPR
system. Giving that,
1. The single strand nick formation can be easily repaired by
HDR cell repair system using the intact complementary DNA
strand as template without leaving any scare.
Cas9 Nickase

35. 2. In order to utilize nickase in gene editing, it
requires two gRNA instead of one. The two gRNA
are designed on opposite DNA strands but with
close proximity to ensure that a DS-blunt ends
are induced once the two strands are nicked by
the Cas9 Nickase.
Cas9 Nickase

36. Cas9 Nickase

37. The two catalytic sites of Cas9 are
deactivated.
Cas9 Double
Mutant
Now Cas9 retains its ability to bind to the
target DNA sequence adjacent to the PAM
sequence through gRNA- genomic DNA
base pairing, but it loses its endonuclease
activity.

38. Cas9 Double
Mutant
 In one example, Cas9 is fused to an enzyme
(deaminase), which mutates specific DNA bases
eventually replacing cytidine with thymidine.
 This precise gene editing means that you could turn a
disease causing mutation into a healthy version of a
gene or introduce a stop codon at specific place.
 Scientists exploit Cas9 ability to specifically binding to
target DNA and fused new enzymes onto the Cas9
protein.

39. Cas9 Double
Mutant
 The same principle applies to the gene silencing. A
KRAP domain fused to Cas9 inactivates transcription by
recruiting more factors that physically block the gene.
This technique is not only used for gene editing
 recently scientists have been working on ways to use CRISPR
to promote gene transcription. They do this by adding
transcription activators to muted Cas9 or recruited to gRNA.
 These activators recruit the cell’s transcription
machinery, bringing RNA polymerase and other
factors to the target and increasing transcription of
that gene

40. Create your
own Gallery
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Cas9Double Mutant

41. saCas9
Nuclease
 A miniature Cas9 nuclease isolated from S. aureus
(saCas9).
 saCas9 is more preferable for in vivo gene editing
over spCas9 because of the following characters;
 It is 1Kb smaller than spCas9 allowing its packaging into
AAV vector. AAV is a preferred method of gene delivery
for in vivo studies due to its low immunogenicity and
ability to selectively infect certain tissue types.
 saCas9 has a different PAM sequence than spCas9
therefore it targets different genomic sites than spCas9,
making it a complementary tool to spCas9.
 As its PAM sequence is longer and rarer in the genome, it
has a reduced risk of off-target cleavage.

42. saCas9
Nuclease
 A study done in 2015 by MIT’s Zhang lab investigated
saCas9 performance in vivo. They injected AAVs
carrying saCas9 into mice to disrupt the PCSK9 gene,
which is linked to familial hypercholesterolemia.
 This resulted in >40% gene modification in liver tissue
after 1 week, with no signs of toxicity 4 weeks after
injection.

43. 43 MASSIVE X presentation to DesignBall team
Cpf1 Nuclease
In late 2015 Feng Zhang’s
group at MIT discovered a new
CRISPR nuclease Cpf1, These
nucleases were found to have
on-target cleavage efficiencies
in human cells comparable
with that of the commonly used
spCas9.

44. Cpf1 Nuclease
1. CPF1 recognizes T-rich PAM sites,
compared to the G-rich PAM of
Cas9, It allows for new targeting
possibilities.
2. Cpf1 may be a useful alternative to
Cas9 in cases where expression
of Cas9 is toxic, as in
Corynebacterium glutamicum and
several species of Cyanobacteria.
3. Cpf1 can process the pre-crRNA
by itself; it does not need
tracrRNA, while Cas9 requires the
presence of a tracrRNA to process
crRNA.
4. Cpf1 requires a shorter guide RNA
to operate. This simplifies the
method, reduce the cost and
makes delivery easier and more
efficient.

45. Cpf1 Nuclease
5. Cas9 generates blunt ends after
cleavage while Cpf1 leaves sticky
5’ overhangs that may be used for
directional cloning, and allows in
vivo gene knock-in in non-
dividing cells as neurons.
6. Cpf1 enables multiple rounds of
DNA cleavage as it cuts DNA 18-
23 bp downstream from the PAM
site, resulting in no disruption to
the recognition sequence and
increased opportunity for the
desired genomic editing to
occur, in contrast to Cas9.

46. spCas9, saCas9,
and Cpf1
nucleases
Comparison between

47. L
S A E
Strength
s
.
Limitation
s
Applications Experimental
variations
CRISPR/Cas9 System

48. CRISPR/Cas9 system has many benefits in terms of
easiness, flexibility, and availability over two other old
genome editing methods ZFN and TALEN.
T
O
W
S
Strength
s
1. CRISPR method depends on the identification of RNA-
DNA instead of the protein-DNA binding process.
2. Creating a modified CRISPR/Cas9 complex is quite
feasible and simpler by merely modifying the gRNA
sequence rather than designing a new protein.
3. CRISPR has great target location specificity, enabling
researchers to make reliable genetic modifications. This
specificity is achieved by CRISPR via the sgRNA.

49. Off-target effects
T
O
L
Limitation
s
 Consequently, attempts were made to enhance the
specificity and prevent the effects of off-target in
genome editing technologies.
 The use of paired nickases dramatically decreases
off-target effects
While the CRISPR system exhibited high specificity in
bacterial cells, when coming to mammalian cells, the system
showed a significantly high frequency of non-specific
nuclease activity thus leading to mutagenesis in regions
other than their particular targets.

50. Applications of CRISPR/Cas9 System
T
A
1. Functional screening of genes
 For elaborate understanding of the functional aspect
of the genome of any model organism by the creation
of a single gRNA library in lentiviral vectors.

 Screening of such libraries was performed by
observing loss of function in knock-out mutants.
 Comparison with the existing RNAi library revealed
reduced off target effects in addition to creation of
knockouts instead of temporary knock down.
Applications

51. Applications of CRISPR/Cas9 System
T
A
2. Creation of cellular and animal models for diagnosis
and gene therapy.
3. Transcriptional studies using fusion proteins and Cas9
Double Mutant (previously discussed)
4. Fluorescence imaging of genome by fusing florescent
protein to Cas9 Double so we can
 see where particular DNA sequences are found in
the cell. This can be useful for visualizing the 3D
architecture of the genome
 to mark the entire chromosome and follow its
position in the nucleus.
Applications

52. Applications of CRISPR/Cas9 System
T
A
5. Antimicrobial and antiviral applications
 Recent studies showed that delivering the CRISPR/Cas9
system designed against the resistance genes of bacteria,
delivered by means of bacteriophages, helped in targeting
the drug resistant candidates thereby making them
sensitive.
 In a similar manner, sequence specific antivirals are also
under consideration, modifying genes involved in host-virus
interaction.
Applications

53. The percentage of the edited
cells can be estimated
E
Mismatch Cleavage Detection Assay(Surveyor
assay).
 In this method, target DNA from the cells is amplified
by PCR then denatured and reannealed, forming
hybrids between unedited and edited strands in the
process.
 Any hybrids with mismatches are then cleaved using
the Surveyor or T7E1 nuclease. Results are run on a
gel to estimate the percentage of edited cells.
 A monoclone must then be isolated and validated,
using Sanger Sequencing to verify the frame shift
mutation.
Experimental
validation

54. Gene
delivery
systems
Viral
Non-viral
Combined
01
02
01
02
01
Lentiviruses
Adenoviral vector
Physical Methods
Chemical Methods
Viorsome: Which is liposome containing inactive HIV
or influenza virus
Gene delivery systems
02
Adeno-associated
vectors

55.  The main types of viral vectors are RNA and
DNA.
 RNA viral vectors involve the use of reverse
transcription to allow the addition to a human
body cell. Ex. retroviral and lentiviral.
 DNA viral vectors directly introduce the genes
to the host genome, such as adenoviral, adeno-
associated, herpes simplex virus, and poxvirus.
Employ viruses to carry DNA into a human cell

56.  Introduce genes to both dividing and non-dividing
cells
 Small-sized
 Few health effects
 High stability.
 Induce minimum immunologic response
Adeno-associated vectors are the scientists favorite,
because;

58.  The physical tools such as gene gun and
microinjection use force to ensure the penetration
of genetic material into a human cell.
 Electroporation is based on the method which
employs high voltage pulse for DNA transfer
through the cell membrane.
 The chemical approaches rely on carriers such as
Liposomes and polymers to send DNA into a human
body. The association of DNA with the polymers is
generally referred to as polyplex.
Facilitate a more effective transfer of DNA
into a human genome through physical
and chemical methods.

59. Cystic Fibrosis
Hemophilia
Growth Rate
Cancer
Countdown step
Diabetes type 1
Gene Therapy for Several
Diseases
Growth Rate
Parkinson’s Disease
Countdown
step

60. Cystic Fibrosis
Since the most mortality rate of CF is for pulmonary
disease the efforts to cure the disease are mostly
concentrated on lungs
Gene therapy has some advantages in comparison to other
drugs; for example,
 it will solve the root of the problem rather than symptoms
 can be used for all CF patients, not just a specific class.
One of the important genes that play a key role in CF gene
therapy is cystic fibrosis trans-membrane conductance
regulator gene (CFTR) that is expressed to lungs epithelial
cells, using viral or non-viral vectors.

61. Hemophilia
 Hemophilia is an X-linked recessive disorder characterized by
bleeding
 There are two approaches we follow in gene therapy for
hemophilia;
1. in vivo-Directly administer the vector which carries our target genes
using AAV vectors
2. Ex vivo-transduced cells and transplantation back to patient using
lentiviral (LV) vectors

62.  One of the most important methods which gene therapy
has given attention to is overexpression of genes.
 Since the reason behind the occurrence of diabetes type
1 is under expression of some specific genes
overexpression of them can be a good choice for gene
therapy, for example
 insulin-like growth factor 1 (IGF1),
 glucose-6-phosphatase (G6Pase) gene,
Diabetes type 1

63. 01
02
03
04
Blocking the expression of an oncogene by
using an antisense (RNA/DNA) approach
Inserting a wild type tumor suppressor gene to
compensate for its loss/deregulation
Expressing a gene to induce apoptosis or enhance tumor
sensitivity to conventional drug/radiation therapy.
Enhancing the immunogenicity of the
tumor to stimulate immune cell recognition
Cancer
There are several prospective strategies
currently being used for targeting cancer
using gene therapy including:

64. Two treatment strategies have been employed in AAV2-
mediated gene therapy clinical trials for PD.
Parkinson’s Disease
1. Symptomatic therapies aimed at improving clinical
symptoms
 Two strategies have been employed, which involved gene
transfer
 Glutamic acid decarboxylase (GAD), which catalyzes
the synthesis of (GABA) from glutamate.
 Aromatic L-amino acid decarboxylase (AADC), a rate-
limiting enzyme for dopamine synthesis that converts
levodopa to dopamine.

65. Two treatment strategies have been employed in AAV2-
mediated gene therapy clinical trials for PD.
Parkinson’s Disease
2. Neurorestorative therapies, focused on attempting to
restore cellular functions affected by the disease process
 have involved in vivo gene transfer of a neurotrophic
factor
 Glial derived neurotropic factor (GDNF)
 Neurturin.

66. Gene Therapy Products on the Market
Strimvelis
Glybera
Gendicine
Luxturna
coronavirus
vaccine

67. Glybera
 Glybera is a gene therapy treatment designed
to reverse (LPLD).
 Lipoprotein lipase deficiency (LPLD) is a rare
inherited disorder which can cause severe
pancreatitis.
 Glybera is composed of an adeno-associated
virus serotype 1 (AAV1) viral vector with an
intact copy of the human lipoprotein lipase
(LPL) gene for delivery to muscle cells.

68. Luxturna
 Luxturna (voretigene neparvovec-rzyl) is
currently the only approved gene therapy
available in the US.
 Luxturna can be used for patients who have
mutations in the RPE65 gene in both
chromosomes, causing retinal dystrophy
 Luxturna is an adeno-associated virus type 2
(AAV2)-based treatment where the correct
copy of the RPE65 gene is delivered without
disturbing the genome.

69. BNT162b2 Pfizer
coronavirus vaccine
 The vaccine uses messenger RNA, genetic material coding
for viral spikes that viral uses to enter human cells.
 By using oily bubbles made of lipid nanoparticles as delivery
vector.
 After injection, the vaccine particles bump into cells and
fuse to them, releasing mRNA. Cells read mRNA and build
spike proteins and presenting them on their surfaces.
 These protruding spikes and spike protein fragments can
then be recognized by the immune system, which mounts
an immune response against these proteins.
 Antibodies against corona virus spikes build up in our bodies
just waiting for the virus to enter blood to deactivate it.

70. Limitations
Name
of Mistakes
1. Gene delivery and activation
2. disrupting important genes in target cells
3. commercial validity
4. financial limitations
5. Somatic gene therapy has had some limited
success.
6. retrovirus-mediated gene transfer resulted
in a potential risk of insertional oncogenesis
The major challenges have been delivery of DNA to the
target cells and duration of expression.
Challenges and Limitations
of Gene Therapy