gene therapy: What worked out and why everthing is not working
1. Gene therapy: what worked out
and why not everything
Department of Medical Biotechnology,
Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University,
Krakow, Poland
Alicja Jozkowicz
2. J.J Bigda & P. Koszałka. Gene 2013
Prof. Wacław Szybalski
McArdle Laboratory
for Cancer Research,
Madison, USA
Gene therapy was born in 1962
Cells
- D98S cells – cell line from human bone marrow:
* with functional HPRT gene (HPRT+)
* without functional HPRTgene (HPRT-)
Medium HAT
- Culture medium that contains:
* Hypoxanthine – source of purines in salvage pathway
* Aminopterin- inhibits synthesis of nucleotides
* Thymidine – source of pirimidine deoxynucleosides
- Only cells expressing HPRT (HPRT+) can survive.
12. Scheme of experiment
pSG5-VEGF
Plasmid harboring
hVEGF165 cDNA
Józkowicz et al. Int J Artif Organs 2003
- VEGF protein level in treated muscle
- Number of capillaries in treated muscle
- Blood perfusion in treated muscle
2 weeks
16. immediately
after ligation
14 days
after ligation
β-gal
VEGF
blood flow in ischemic adductor muscles
blood
flow
in
muscle
(100%
-
blood
flow
before
ischemia)
0
20
40
60
80
100
120
β-gal hVEGF
*
Effects of hVEGF plasmid delivery
Józkowicz et al. Int J Artif Organs 2003
17. First clinical delivery of VEGF plasmid
Improvement was not sufficient to salvage leg.
Amputation was performed 5 months after gene delivery.
- Patient: 71-old woman with ischemic leg
- Treatment: plasmid phVEGF165 (2 mg) was
applied to the hydrogel polymer coating of an
angioplasty balloon and delivered to the
popliteal artery.
- Effects: increase in collateral vessels, increase
in vascularization, increased resting and
maximal blood flow
- Side effects: Three spider angiomas developed
on treated foot - one was excised, other two
regressed. The patient developed oedema in her
right leg, which was treated successfully.
Isner et al. Lancet 1996
18. Baumgartner et al. Circulation, 1998
Clinical trial: delivery of VEGF plasmid
- 9 patients with nonhealing ischemic
ulcers due to peripheral arterial
disease.
- 4 mg of naked plasmid with hVEGF
injected into the muscle.
before treatment 8 weeks
after treatment
before 4 weeks 8 weeks
ABI
angiograms
19. Baumgartner et al. Circulation, 1998
Limb salvage after gene therapy
- A 33-year-old woman, wheelchair-bound on multiple analgesics.
- Nonhealing wound on calf and ischemic
necrosis involving great toe.
- Ingrowth of granulation tissue in calf wound,
healing of great toe.
- Three months after gene transfer, healing of
split-thickness skin graft at wound site and full
resolution of great toe necrosis.
20. Kastrup et al. JACC 2005
Placebo-controlled trial
For example:
- 80 patients with stable severe angina pectoris, in experimental and placebo
groups, treated with VEGF cDNA delivery using plasmid (The Euroinject One Trial)
Results of placebo-controlled trials are elusive. Plasmid-mediated VEGF therapy is
safe but only marginally effective in patients with angina pectoris.
voltage
baseline
(mV)
voltage at
3 months
(mV)
LLS at
3 months
(%)
LLS
baseline
(%)
myocardial perfusion myocardial perfusion
placebo
VEGF
21. Stewart et al. Mol Ther. 2009
Placebo-controlled trial
For example:
- 93 patients with advanced coronary disease, in experimental and placebo groups,
treated with VEGF cDNA delivery using plasmid NORTHERN Trial)
Placebo-controlled trials do not support the use of therapy with the VEGF in people
with ACD to improve myocardial perfusion and walking ability.
exercise treamill time
22. Miao et al. Vasc Endovasc Surgery 2014
Placebo-controlled trial: meta-analysis
Also: no differences (P=0.23) in serious adverse effects
major amputation or death at 1 year complete wound healing at 6 months
P=0.48 P=0.25
Conclusion: lack of beneficial effects in critical limb ischemia
24. Kusumanto et al. Hum Gene Ther. 2006
Placebo-controlled trial
For example:
- 54 patients with critical limb ischemia, in experimental and placebo groups,
treated with VEGF cDNA delivery using plasmid vectors.
clinical effects
Placebo-controlled trial show some positive effects the use of therapy with the
VEGF in diabetic patients with CLI in improvement of skin ulcer healing.
25. Deev et al. J Cardiovasc Pharmacol Therapeutics 2015; Hanna et al. J Market Access Health Policy 2017.
Vectors used for gene delivery
Neovasculgen, plasmid coding VEGF, for critical hind-limb ischemia
manufactured by Human Stem Cells Institute, Moscow
approved by Russian Ministry of Healthcare in 2011
before treatment 6 months after vegf165
gene transfer.
26. Deev et al. J Cardiovasc Pharmacol Therapeutics 2015; Hanna et al. J Market Access Health Policy 2017.
Vectors used for gene delivery
Neovasculgen, plasmid coding VEGF, for critical hind-limb ischemia
manufactured by Human Stem Cells Institute, Moscow
approved by Russian Ministry of Healthcare in 2011
Inhibition of angiogenesis:
aptamers
before treatment 6 months after vegf165
gene transfer.
27. Histological organization of retina
In humans, a circular avascular zone of about 450 µm at the fovea
improves central vision by reducing light scatter from blood vessels.
Gariano and Gardner, Nature 2005
29. - Pegaptanib (Macugen): covalent conjugate of an oligonucleotide of 28-nucleotides
combined with two 20-kD monomethoxy polyethylene glycol (PEG).
- Comprises natural ribonucleotides as well as not natural nucleotides, with 2′-fluoro
or methoxy groups.
Macugen: anti-VEGF aptamer
Dunn et al. Nature Rev Chem 2017
30. Macugen (aptamer blocking VEGF) –
first anti-angiogenic drug approved for
treatment of ophthalmological diseases
Macugen: anti VEGF aptamer
Inhibition of VEGF binding to the receptors in cells treated with macugen
VEGFR1 VEGFR2
binding
[a.u.]
binding
[a.u.]
Dunn et al. Nature Rev Chem 2017
33. https://uctclinic.com/spinal-muscular-atrophy; SJ & Kissel JT. Neurological Review 2011.
Spinal muscular atrophy (SMA)
Spinal muscular atrophy (SMA)
- a neurodegenerative disease characterized by loss of motor neurons in the
anterior horn of the spinal cord and resultant weakness.
34. Wirth et al. Ann Rev Genomics Hum Dis 2020.
Spinal muscular atrophy (SMA)
SMN1 and SMN2: from the gene to protein
The difference: a translationally silent mutation at exon 7, which is part of an
exonic splicing enhancer in SMN1 (blue box with a C) and an exonic splicing silencer
in SMN2 (red box with a T ).
35. Wirth et al. Ann Rev Genomics Hum Dis 2020.
Spinal muscular atrophy (SMA)
Nusinersen-mediated restoration of SMN2 splicing
Nusinersen (black triangles) blocks the splicing site (red triangles), impairing the
access of splicing factors. It thereby promotes exon 7 inclusion in SMN2 mature
transcripts and shifts the balance toward increased full-length SMN protein levels.
~10,000 patients
38. Adenoviral life cycle
Entry into the cell
Uncoating of DNA
replication
transcription
translation
Assembly of progeny
virus particles
and exit from cell
DNA
RNA
39. Adenoviral vector action
Entry into the cell
Uncoating of DNA
replication
transcription
translation
RNA
Therapeutic protein
produced from transgene
40. Adenoviruses
- App. 50 serotypes of adenoviruses causing mild illnesses in humans
- Genome: 36 kb double-stranded linear DNA consisting:
* Early genes (for viral gene transcription and DNA replication)
* Late genes (coding proteins required for virus assembly)
- E1 early genes: essential for the subsequent adenoviral gene expression
E1
ITR ψ+
ITR
transgene
ITR ψ+
ITR
41. Production of ∆E1 adenoviral vectors
transgene
ITR ITR
HEK293 cells (stably transfected with E1 region)
E1 E1 E1 E1 E1
43. Suzuki et al. FEBS Lett 1998, Soudais et al. J Virol 2000
Adenoviral vectors preferentially target the liver after intravenous injection
Adenoviral vectors: in-vivo efficacy
Naked plasmid Adenoviral vector
Hepatocytes, GFP
44. Immune response to adenoviral vectors
Elkon et al. Proc Natl Acad Sci USA 1997, Jay et al. Blood 1999
control
low dose Ad
high dose Ad
Ad-injection control
Liver crossection (H&E staining)
45. Immune response to adenoviral vectors
Lehrman S. Nature 1999
September 1999
Researchers at the University of Pennsylvania's
Institute for Human Gene Therapy used an E1-
deleted adenoviral vector to transduce liver cells
with the correct ornithine transcarbamylase OTC
gene, which codes for a urea-cycle enzyme that
removes excess nitrogen from the body.
18-year-old Jessie Gelsinger, the eighteenth and
final patient in the Phase I experiment, was the
second person to receive a dose of 3.8×1013 viral
particles.
He developed a fever and blood clots throughout
his body within hours of treatment. He died four
days later.
Jessie Gelsinger
46. Adenoviral vectors of first generation
Great:
- Very high transduction efficiency
- Broad host and cell type ranges
- Can be prepared in high titers
- Can transduce mitotic and post-mitotic cells
- Do not integrate with genome
- Can harbor ~ 7 kb of transgene
But:
- Strong immune response to viral proteins eliminates transduced cells
- Neutralizing antibody prevents readministration of vector of the same serotype
Thus:
Adenoviral vectors provide the high but transient transgene expression
47. Adenoviral vectors of first generation
Great:
- Very high transduction efficiency
- Broad host and cell type ranges
- Can be prepared in high titers
- Can transduce mitotic and post-mitotic cells
- Do not integrate with genome
- Can harbor ~ 7 kb of transgene
But:
- Strong immune response to viral proteins eliminates transduced cells
- Neutralizing antibody prevents readministration of vector of the same serotype
Thus:
Adenoviral vectors provide the high but transient transgene expression
Making adeno-vectors safer:
gutless adenoviral vectors
50. Structure of HDAd-apoE
Kim, Jozkowicz et al. PNAS 2001
HD-AdgenApoE (33.4 kb)
ITR ψ+
ITR
LSE
⇐ ⇒
APOEpr
ApoE
(exons and introns)
ApoE polyA
44 66 193 869
Scheme of ApoE gene
apoE cholesterol atherosclerosis
51. Transgene expression in mice
ApoE protein in blood of ApoE KO mice injected
with ApoE adenoviral vectors
(western blot, 4 weeks after injection)
AdE1-apoE HdAd-PEPCK-
apoE
PBS
HdAd wild
mouse
HdAd-gen-
apoE
Kim, Jozkowicz et al. PNAS 2001
52. Plasma ALT after adenoviral vector injection
Weeks after treatment
0 1 2 3 4 5 6 7
ALT
(IU/L)
0
100
200
300
400
500
600
AdE1-apoE
HdAd-gen-apoE
PBS (control)
**
*
Kim, Jozkowicz et al. PNAS 2001
53. Treatment efficacy in mice
ApoE level in the blood of mice
after single injection with apoE gutless vector
0 112 224 336 448 560 627 784
Plasma
ApoE
[mg/dl]
2
4
6
8
10
0
AdE1-apoE
HdAd-PEPCK-apoE
HdAd-gen-apoE
Days after injection
Kim, Jozkowicz et al. PNAS 2001
54. Aortas and mice stained with oil red
817 days after injection with apoE gutless vector
apoE-/- control HdAd-gen-apoE
Treatment efficacy in mice
Kim, Jozkowicz et al. PNAS 2001
55. - Very high transduction efficiency
- Broad host species and cell type range
- Can transduce mitotic and post-mitotic cells
- Can harbor ~ 35 kb (!) of transgene
- Do not integrate with genome
- Do not produce any viral proteins
- Show significantly reduced immunogenicity in-vivo
Drawback:
- Difficult for production in high titers
- Recognized by neutralizing antibodies
- Can iduce strong immune response to the viral capsid proteins
Gutless adenoviral vectors
56. - Very high transduction efficiency
- Broad host species and cell type range
- Can transduce mitotic and post-mitotic cells
- Can harbor ~ 35 kb (!) of transgene
- Do not integrate with genome
- Do not produce any viral proteins
- Show significantly reduced immunogenicity in-vivo
Drawback:
- Difficult for production in high titers
- Recognized by neutralizing antibodies
- Can iduce strong immune response to the viral capsid proteins
Gutless adenoviral vectors
Naturally safer: AAV vectors
58. AAV vectors
- Transduce different cells, both proliferating and not proliferating.
- They do not induce strong inflammatory response and can sustain
relatively long-term expression.
- Remain as episomal particles (despite capability of wild-type AAV to
intergrate in a strictly defined site on chromosome 19).
- Low capacity: usually ~4.5 kb
F
. Mingozzi, K.A. High, Blood 2013
AAV
Ad
60. Hanna et al. J Market Access Health Poliicy 2017.
Vectors used for gene delivery
Glybera (AAV1)
AAV-vector coding LPL, for dyslipidemia caused lipoprotein lipase deficiency
- manufactured by UniQure,
- approved as orphan drug by EMEA in 2012,
- not prolonged in 2017 (one patient was treated)
61. AAV: treatment of hemophilia A
Rangarajan et al. NEJM 2017
single infusion, 9 patients
before after after
effective
Annualized bleeding rate
before after after
effective
Annualized factor VIII use
63. MacLaren et al. Lancet 2014.
Choroideremia (REP1 deficiency)
- Progressive loss of vision due to
degeneration of the pigmented epithelium
and then degeneration of the retina and
choroid.
- Night blindness is followed by loss of
peripheral vision (tunnel vision) and later a
loss of central vision.
64. MacLaren et al. Lancet 2014.
Choroideremia (REP1 deficiency)
Vector doses and vision changes (after 6 months) in patients with
choroideremia
Rep1
tubulin
65. Retinitis pigmentosa (RPE65 deficiency)
The RPE65 protein is located in the retinal pigment epithelial (RPE) cells and
converts all-trans-retinol to 11-cis-retinol, which subsequently forms 11-cis-retinal
during the visual cycle.
The RPE65 is 1 out of more than
270 genes that may be responsible
for inherited retinal disease.
Russel et al. Lancet 2017.
66. Russel et al. Lancet 2017.
Voretigene, AAV-vector coding RPE65, for vision loss
manufactured by Spark
approved as: orphan drug by FDA in 2017, priority by FDA and priority by EMEA
Retinitis pigmentosa (RPE65 deficiency)
67. Al-Zaidy SA & Mendell JR. Pediat Nerol 2019.
Spinal muscular atrophy (SMA)
Spinal muscular atrophy (SMA)
- a neurodegenerative disease characterized
by loss of motor neurons in the anterior horn
of the spinal cord and resultant weakness.
- The most common form of SMA (95% of cases), is autosomal recessive proximal
SMA associated with mutations in the survival of motor neurons (SMN1) gene.
Nusinersen (antisense oligos) Zolgensma (AAV gene therapy)
69. p53 protein is often inactive
in cancer cells
DNA quality control: p53
Li et al. BMC Medicine 2014
70. Ad-p53 in anticancer therapy
- 99 patients with stage III or IV oral carcinoma (ineligible for surgery) were enrolled
in a randomized, placebo-controlled, double-blind, phase III clinical trial:
* group I: intra-arterial infusion of rAd-p53 plus chemotherapy
* group II: intra-arterial infusion of rAd-p53 plus placebo chemotherapy
* group III: intra-arterial infusion of placebo rAd-p53 plus chemotherapy.
Li et al. BMC Medicine 2014
*
group II
group I
group III
stage III
group II
group I
group III
NS
stage IV
Gendicine (SiBiono GeneTech, China, registered in 2003)
71. Hanna et al. J Market Access Health Poliicy 2017.
Gencidine, adeno-vector coding p53, for head and neck cancer
manufactured by Shenzen Sibiono GeneTech
approved by Chinese State Food and Drug Agency in 2003
Ad-p53 in anticancer therapy
72. Ad-p53 in anticancer therapy
- >12 years of commercial use in >30,000
patients, >30 published clinical studies
- good safety record (no serious adverse
events have been reported, except for
vector-associated transient fever in 50–60%
of patients)
- when combined with chemotherapy and
radiotherapy: higher response rates than
for standard therapies alone
- p53 mutation status did not significantly
influence efficacy outcomes and long-term
survival rate for Ad-p53-treated patients
Yuan et al. SpringerPlus 2016; Zhang et al. Human Gene Ther 2018
complete remission rate of primary tumors
Gendicine (SiBiono GeneTech, China, registered in 2003)
73. Ad-p53 in anticancer therapy
- >12 years of commercial use in >30,000
patients, >30 published clinical studies
- good safety record (no serious adverse
events have been reported, except for
vector-associated transient fever in 50–60%
of patients)
- when combined with chemotherapy and
radiotherapy: higher response rates than
for standard therapies alone
- p53 mutation status did not significantly
influence efficacy outcomes and long-term
survival rate for Ad-p53-treated patients
Yuan et al. SpringerPlus 2016; Zhang et al. Human Gene Ther 2018
complete remission rate of primary tumors
Gendicine (SiBiono GeneTech, China, registered in 2003)
New challenge: SARS-CoV-2
74. Falsey et al. NEJM 2021.
Ad-vector delivered vaccine
local adverse effects
systemic adverse effects
75. Falsey et al. NEJM 2021.
Time to first SARS-CoV-2 RT-PCR–positive symptomatic illness
occurring 15 days or more after the second dose
32,451 participants
Ad-vector delivered vaccine
