Gene therapy is a medical technology which aims to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells.

1. Presented by:
Mostafa Changaei
School of Medical Sciences
Department of Immunology
An Overview on Gene Therapy
Platforms
30 Ord 1402
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2. 2/37

3. 3/37
Evolution of Medical Technologies
From “Jin” Therapy to “Gene” Therapy
Body Organ Cell Gene

4. Gene Therapy Definition
Gene therapy is a technique that modifies a person's genes to treat or cure disease
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Central Dogma of Biology

5. 1970
Gene therapy
Concept established
1990
First clinical trial to deliver
a therapeutic gene
(ex vivo retroviral vector)
2003
China approved recombinant
human p53 adenovirus
(in vivo adenoviral vector
for treatment of HNSCC)
2012
First gene therapy
approved in the Eu,
for treatment of patients
with familial LPLD
(in vivo AAV vector)
2017
August
First CAR T-cell therapy
approved by FDA for ALL
(ex vivo lentiviral vector)
October
FDA approved gene therapy
for large B-cell lymphoma
(ex vivo retroviral vector)
December
First rAAV product approved
for treatment of bi-allelic RPE65 gene
mutation-associated retinal dystrophy
(ex vivo retroviral vector)
2019
May 24
First gene therapy approved in the US
for treatment of SMA
(in vivo AAV vector)
May 29
First gene therapy approved
for treatment of
transfusion-dependent β-thalassemia
(ex vivo retroviral vector)
2016
First ex vivo gene
therapy for
treatment of ADA-SCID
was approved
(retroviral vector)
2018
FDA approved ex vivo gene
therapy for DLBCL
2008
First adenovirus-based
gene therapy intended for
treatment of malignant
brain tumors
1999
Death of a clinical
trial participant due
to severe immune
reaction following
in vivo adenoviral vector
administration
1989
First approved
clinical trial protocol
to use gene transfer
into human
History of Gene Therapy: The Major Milestones
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6. Gene Therapy Approaches
Gene Editing Vectors
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7. Gene Therapy Approaches
CRISPR-Cas 9 Viral Non-Viral
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Gene Editing Vectors

8. Genome Engineering Using the CRISPR-Cas9 System
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9. Genome Engineering Using the CRISPR-Cas9 System
 Cas9 promotes genome editing by stimulating a
double strand break (DSB) at a target genomic locus
 CRISPR-Cas is a microbial adaptive immune system
that uses RNA-guided nucleases to cleave foreign
genetic elements.
 Cas9 is a nuclease guided by small RNAs through
Watson-Crick base pairing with target DNA.
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10. DSB Repair Promotes Gene Editing
NHEJ; Non-homologous end joining HDR; Homology-directed repair gRNA; Guide RNA
Scenario 1 Scenario 2
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11. Inducing a Rat Model for SCID
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12. Knockout of the Il2rg and Rag2 Genes
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13. Knockout of the Il2rg and Rag2 Genes
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14. A
Flow Cytometry Analysis of Cell Populations in Peripheral Blood
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15. A
Flow Cytometry Analysis of Cell Populations in Peripheral Blood
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16. A
Flow Cytometry Analysis of Cell Populations in Peripheral Blood
A: Analyses of SSC and CD3
Dot plots show the distributions of the cells after lymphoid gating. The SSC
low/CD3+ T cell group is indicated in the box as SSCloCD3+.
Q1 Q2
Q3 Q4
IL-2R KO IL-2R KO
WT (Ctrl) 16/37

17. A
Flow Cytometry Analysis of Cell Populations in Peripheral Blood
B: Analyses of CD4 and CD8
Dot plots show the distributions of CD3+ cells.
IL-2R KO IL-2R KO
WT (Ctrl)
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18. A
Flow Cytometry Analysis of Cell Populations in Peripheral Blood
C: Analyses of CD45RAand CD161a
Dot plots show the distributions of CD3+ cells. (C) Analyses of
CD45RA and CD161a. Cells from the SSCloCD3− gate were
divided into groups. CD3−/CD45RA+ B cells and CD3−/CD161a+
NK cells are shown in the boxes
IL-2R KO IL-2R KO
WT (Ctrl)
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19. Work Flow of CRISPR/Cas Gene Editing in Gene Therapy of IEIS
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20. The FOXP3 locus is targeted using the CRISPR system in primary HSPCs and T cells
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21. The FOXP3 locus is targeted using the CRISPR system in primary HSPCs and T cells
Poly A Sequence
Phosphoglycerate Kinase Promoter
NGFR (NGFR) Marker Gene
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22. T reg Marker Expression
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23. Marianne Goodwin
2020
CRISPR-based gene editing enables FOXP3 gene repair in IPEX
patient cells
M. Goodwin1*, E. Lee1,2*, U. Lakshmanan1, S. Shipp1, L. Froessl1, F. Barzaghi3,
L. Passerini3, M. Narula1, A. Sheikali1, C. M. Lee4, G. Bao5, C. S. Bauer6, H. K.
Miller6, M. Garcia-Lloret7, M. J. Butte7, A. Bertaina1, A. Shah1, M. Pavel-Dinu1,
A. Hendel1,8, M. Porteus1,2, M. G. Roncarolo1,2, R. Bacchetta
S C I E N C E A D V A N C E S Immunology
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24. Work Flow of CRISPR/Cas Gene Editing in Gene Therapy of IEIS
Autologous HSCs collected from patients undergo ex vivo
culture and CRISPR/Cas editing, after screening and expansion,
therapeutic edited cells are transfusion into conditioned patients
for immune system reconstruction. CRISPR/Cas gene editing
agents could be delivered into HSCs in forms of RNP, “all
RNA”, or AAV vector to enable efficient pathogenic gene
correction.
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25. Gene Editing Vectors
CRISPR-Cas 9 Viral Non-Viral
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26. Two Major Classes of Mobile Elements
Cut & Paste Copy & Paste
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27. Viral Vectors Used in Gene Therapy
Adenovirus Vector Adeno-associated Vector
(AAV)
Lentivirus Vector
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28. Viral Vectors Used in Gene Therapy
Lentivirus Vector
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29. Lentiviral Vectors
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30. Adenovirus Vector
Adeno-associated Vector
(AAV)
Viral Vectors Used in Gene Therapy
 Inverted-terminal repeats (ITRs) serve as self-priming structures that promote primase-independent
DNA replication.
 The early-phase (E1-E4) genes are transcribed before the initiation of viral DNA replication.
 The “late-phase” genes (L1–L5) are generally required for virus assembly, release, and lysis of the
host cell.
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31. Viral Vectors Used in Gene Therapy
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32. Comparison Between Viral Vectors
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34. Viral Vectors Used in Gene Therapy
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35. Summary
Gene therapy: is the treatment of a genetic disease by the introduction of specific cell function-altering genetic
material into a patient.
Gene Editing Viral Vectors
Lentiviral Adeno/AAV
Not-integrating
Integrating 35/37

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38. Future Concerns
Horizontal Gene Transfer
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Economist

39. Thanks for Y
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41. Adenoviral & AAV Vectors
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43. Adenoviral & AAV Vectors
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44. 2) Great packaging capacity (up to 8kb).
3) Broad range of infectivity. Adenovirus can infect both
dividing and quiescent cells, allowing gene delivery to a
highly diverse range of cell types.
4) It can be produced at high titer (10^10 VP/mL, which
can be concentrated up to 10^13 VP/mL).
5) High infection efficiency. Almost 100% gene delivery in
most cell types, completely surpassing other viral vector
tools and liposome transfection.
6) Without integration into the host chromosome.
Adenovirus remains epichromosomal in cells and does not
inactivate genes or activate oncogenes.
b) Drawbacks of Adenovirus-mediated gene transfer
Although adenovirus benefits a great deal of disease
therapies, it does present some drawbacks.
1) Adenovirus-mediated gene delivery may not sustain for
long time, just transient expression.
2) Generation of neutralizing antibodies against
adenovirus in the Non-Human Primates (NHP) and
human, may attenuate the cure effect of adenovirus-
mediated gene therapy [13].
3) Adenovirus vector infection can activate a wide variety
of immune responses both humoral and cellular, which
may increase the risk factor to use adenovirus as vectors
in that high dose will result in acute toxicity and 44/37

45. Adeno Associated Virus (AAV) a) Advantages of AAV-
mediated gene transfer
AAV has been developed into a very attractive candidate
for creating viral vectors for gene therapy and the creation
of isogenic human disease models due to various
advantages. 1) Superior biosafety rating. The wild type
AAV has not currently been known to cause disease in
vivo, and further security of recombinant AAV gene
delivery in vivo is ensured after removal of most AAV
genome elements. 2) Low immunogenicity. AAV causes a
very mild immune response in vivo, lending further
support to its apparent lack of pathogenicity during gene
delivery. 3) Broad range of infectivity. AAV can infect both
dividing and quiescent cells in vivo, allowing gene delivery
to a highly diverse range of cell types. 4) Stable
expression. Long term gene delivery in vivo can be
mediated by AAV. b) Drawbacks of AAV-mediated gene
transfer
Although adenovirus benefits a great deal of disease
therapies, it does present some drawbacks.
1) The major drawback is its limited cloning capacity (less
than 4.7kb) of the vector, which restricts its use in gene
delivery of large genes.(Table 3) [32]. 2) Generation of
neutralizing antibodies against AAV in the Non-Human
Primates (NHP) and human, may attenuate the cure effect 45/37

46. a) Advantages of lentivirus -mediated gene delivery
Lentivirus has been developed as an attractive candidate
for creating viral vectors for gene therapy due to various
advantages.
1) Customized cloning for any other gene ORF
expression, shRNA/miRNA and CRISPR/Cas9.
2) No known immunogenic proteins generated.
3) High titer. 108TU/ml or 109TU/ml lentiviral titer for cell
line transfection in medium or large scale.
4) With broad range of hosts. Mediate efficient
transfection in both dividing and non-dividing cells.
5) Integration into host cell genome, mediating long-term
and stable expression of exogenous genes.
6) Deliver complex genetic elements, such as intron-
containing sequences.
7) Simple system for vector manipulation and production.
b) Drawbacks of lentivirus-mediated gene transfer
Although lentivirus benefits a great deal of disease
therapies, it does present some drawbacks.
1) Based on HIV-1, recombinant lentivirus vectors require
at least three HIV-1 genes (gag, pol, and rev) for
production, which is still not safe enough for gene therapy.
To date, the best solution for this drawback is to turn to
adenovirus or AAV vectors, which may be safer than
lentivirus vector. 46/37

47. Lentiviral Vectors
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53. Summary of viral gene therapy modalities. In vivo gene
therapy entails the direct administration of vector carrying
a therapeutic
transgene into the patient. Ex vivo gene therapy involves
the extraction of a patient’s cells or from an allogenic
source, genetic modification
by a vector carrying a therapeutic transgene, selection
and expansion in culture, and infusion to re-introduce the
engineered cells back into
the patient
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54. polymerase (Pol). The E4 gene encodes 1–7 open
reading frames. The major late messenger RNAs (L1–L5)
mainly encode for virion structural
proteins and are derived from a pre-mRNA, which is
driven by a major late promoter (MLP) via alternative
splicing and polyadenylation. L1
encodes for IIIa and 52K. L2 encodes for the penton base
gene (capsid protein III) and the core proteins V, pVII, and
pX. L3 encodes for the
hexon (capsid protein II), capsid protein precursor (pVI),
and protease (Pr) genes. L4 encodes for 100K, 33K, 22K,
and pVIII. L5 encodes for the
fiber gene (capsid protein IV). b–e Diagrams of rAd
vectors. b Replication-defective (RD) vector. The E1A and
E1B regions are deleted and
replaced with an expression cassette containing an
exogenous promoter and a transgene of interest
(indicated by the solid red X and yellow
arrow). The E3 and E4 regions are usually deleted to
accommodate larger insertions and eliminates leaky
expression of other viral genes.
c E1B-55K replication-competent vector. The E1B-55K
region is deleted (solid red X and dashed blue arrow),
whereas in some vectors, the E3
region is deleted and replaced with an expression 54/37

55. Fig. 4 Schematic of the AAV genome and sites used for
PCR screening. The AAV genome comprised four known
open reading frames, rep
(green), cap (salmon), MAAP (orange), and AAP (yellow).
The rep and cap ORFs encode four and three isoforms,
respectively. Transcription is
driven by the viral P5, P19, and P40 promoters (arrows).
The genome is flanked by inverted terminal repeat (ITR,
cyan) sequences
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56. Third-generation HIV-1-based lentiviral vector design. The
third generation of lentiviral vectors are produced using
four plasmids.
The first plasmid has a construct carrying the gene of
interest driven by a desired promoter flanked by long
terminal repeats (LTRs). Both 5′
and 3′ LTRs in wild-type HIV-1 is composed of U3, R, and
U5 sequences. The U3 sequence constitutes
promoter/enhancer elements. Part of the
U3 sequence in the 3′-LTR is deleted, and the entire U3
sequence within the 5′-LTR is replaced by a strong viral
promoter, such as CMV. The psi
(ψ) packaging signal is followed by the rev response
element (RRE). The envelope glycoprotein is encoded by
VSV-G (vesicular stomatitis virus)
and is expressed under a strong promoter, such as CMV.
The rev gene is split from the packaging plasmid and is
provided on a separate
plasmid construct. The packaging plasmid harbors the
viral gag and pol genes, and notably lacks the tat
regulatory gene
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58. using non-viral
vectors. Various non-viral vectors can be used to deliver
DNA, mRNA and short
double-stranded RNA, including small interfering RNA
(siRNA) and microRNA (miRNA)
mimics. These vectors need to prevent degradation by
serum endonucleases and
evade immune detection (which could be achieved by
chemical modifications of
nucleic acids and encapsulation of vectors). They also
need to avoid renal clearance
from the blood and prevent nonspecific interactions (using
polyethylene glycol (PEG)
or through specific characteristics of particles). Moreover,
these vectors need to
extravasate from the bloodstream to reach target tissues
(which requires certain
characteristics of particles and specific ligands), and
mediate cell entry and endosomal
escape (by specific ligands and key components of
carriers). siRNA and miRNA mimics
must be loaded into the RNA-induced silencing complex
(RISC), whereas mRNA must
bind to the translational machinery. DNA has to be further
transported to the nucleus 58/37

59. and a
cationic lipid. Cationic lipids (such as DOTMA, DOSPA, DOTAP,
DMRIE and
DC-cholesterol) have an active role in DNA binding and
transfection. They
are characterized structurally by a cationic head group, a
hydrophobic tail
and a linker region. Neutral lipids (such as the phospholipids DSPC
and
DOPE, and the membrane component cholesterol) function as
‘helper
lipids’ to further enhance nanoparticle stability and overall
transfection
efficacy. b | Chemical structures of selected polymeric DNA vectors
that are
commonly used in gene delivery studies and clinical trials are
shown.
Poly(l-lysine) and polyethylenimine (PEI) are among the oldest and
most
commonly used polymeric gene vectors. To improve safety and
efficacy,
numerous other polymers have been studied for gene delivery,
including
methacrylate-based polymers such as poly[(2-dimethylamino)
ethyl methacrylate] (pDMAEMA), carbohydrate-based polymers 59/37

60. payload from the endosomal compartment. b | A Cyclodextrin
polymer (CDP)-based
nanoparticle is shown. CDPs are synthesized through
polymerization of
diamine-bearing cyclodextrin (dark green) and dimethyl
suberimidate, which yields
oligomers (n ~ 5) with amidine groups (light blue)35. The
positively charged amidine
groups interact with nucleic acids to form stable particles. The
polymers are
end-capped with imidazole functional groups, which have been
shown to facilitate
endosomal escape154 and improve delivery efficacy of small
interfering RNAs (siRNAs)125.
Adamantane (AD) is a hydrophobic molecule that forms a
stable inclusion complex with
the cyclic core of cyclodextrin155. CDP–siRNA nanoparticles are
formulated with PEG
(the molecular mass of which is 5,000) for stability and long
circulation time, as well as
with transferrin (Tf), which induces the uptake by cells
expressing the transferrin
receptor155,156. c | A Dynamic PolyConjugate (DPC) is shown.
DPCs are composed of a
membrane-destabilizing polymer PBAVE, the activity of which is 60/37