Recombinant virus techniques

(For Gene transfer and Gene Therapy)

徐松錕 (Song-Kun Shyue )

Institute of Biomedical Science, Academia Sinica
中央研究院 生物醫學科學研究所
skshyue@ibms.sinica.edu.tw; Tel: 2652-3962
 References
1. Understanding gene therapy / N.R. Lemoine, New York,
N.Y. : BIOS Scientific Publishers, 1999
2. 生技公司網站：Clontech, Bio-Rad, Stratagene.
3. Annu. Rev. Med. 2005. 56:585–602
4. Mol Ther. 2005 May;11(5):661-76.
5. TRENDS in Biotechnology. 2003. 21(3):117-122.
6. Nature Reviews Genetics. May 2003. 4:346-358.
7. http://www.wiley.co.uk/wileychi/genmed/clinical/
8. Hendrie PC, Russell DW. Gene Targeting with Viral
Vectors. Mol Ther 2005.12(1):9-17.
 Gene Therapy and Transfer
• Deliver therapeutic genetic material to target site
or cells
• Somatic gene therapy
– Introduction a functional gene into somatic
cells in order to restore a defective gene copy
or express protein for the treatment of disease
– Limited to severe diseases
• High efficiency for cellular gene transfer (viral
vector)
 Methods for Gene Transfer

Non-viral vectors
• Naked DNA
• Liposome: Lipid/DNA complex
Viral vectors
• RNA virus:
– Retrovirus, Lentivirus (HIV)
• DNA virus: Adenovirus, Adeno-associated
virus (AAV), HSV-1
• Other viral vectors
 You should know from this lecture

• Understand the design of viral vector

• Properties of common viral vectors
• How to prepare?
• Can we use it?
• Recent progress of gene therapy:
– Good and bad of using viral vectors
– Clinical trial and associated problems
Ex Vivo In Vivo

Patient
 Naked DNA
• Purified DNA (Unlimited size)
– hydro-gel (very low efficiency): Report to improve
blood supply the ischaemic limb with pVEGF (Isner et al.,
1996)

– Stopped blood flow (high efficient in specific area,

need to be further studied)
• Gene gun
• DNA vaccine, with electric pulse
• Limited delivery area (e.g., vascular endothelium)
 Gene Gun

•Helios Gene Gun

•DNA or RNA and gold
carriers
•Require small amount of DNA
•Gene therapy
•DNA vaccination
 Liposomes
• Cationic liposome/DNA complex
• Controlled toxicity
• Unlimited DNA size
• Very low integration
• Very low transfected efficient, below 1%
• Various properties (thermo- or pH-sensitive,
Immunoliposome, etc.)
• Cancer gene therapy human trails
 Good and Bad of Viral Vectors
Advantages
• High efficiency
• Replication deficient viral vectors available
• High titer preparation is possible for many vectors
• Longer expression period with DNA integration, 2nd
generation Ad and helper dependent Ad.
Disadvantages
• May exert immune response against viral vector or
viral transfected cells, second administration limited.
• Possible of integration mutation (AAV and retrovirus),
risk of cancer or other genetic diseases.
AAV
 Terminology
• Plaque forming unit: pfu
– Plaque
– Antibody
– TCID50
• Infectious unit and particle unit
• MOI: Multiplicity of infection
– Plaque per cell
– Particle number per cell
– Infection unit per cell
 Viral Vectors
• Retrovirus (~ 10 kb)
• Lentivirus (HIV base, ~ 10 kb)
• Adenovirus (5, 7, or 35 kb)
• Adeno-Associated Virus (~ 5kb)
• Herpes Simplex Virus-1 (human)
• SV40, Sandi Virus, and other viruses
 Adenovirus
• Well studied (1970s)
• Double stranded DNA ~36 kb, 70~100 nm
• More than 51 characterized serotypes
• Ad5 and Ad2 are less oncogenic; less safety concerns
• ~100% seropositive of Ad5 in Taiwan,
• Infect both dividing and nondividing cells
• High transfer efficiency in a wide variety of cell types
and tissues;
• Very high titer: up to 10E12 PFU/ml or higher
• Large-scale production available
• Low integration frequency,10E-6, in cell culture
 Late genes
pTP ψ ITR

POL ITR E1 E2 E3 E4
ssDBP

E1

Late gene Packaging

expression
 pJM17
• Circular genomic DNA of dl309 (Ad5)
– With small deletion within E3
– Contains only an XbaI site for cloning
– Contains an 100/0 (tail-to-head) junction, will be
processed into a linear viral DNA in cell, may due to
the function of terminal protein
• Clone into pBRX:
– Contains an XbaI
• Total ~ 40kb
Recombinant Ad construction
(First-generation, E1 and/or E3 deleted)
100/0
ϕ pBRX (0~1.3%)
ITR Ad5 (9.25%-16%)
p cDNA pA
E1 ϕ

pAd-pgk-cDNA
pJM17
pBR322

pBR322

293 cells (0% ~ 12% of Ad5);

Homologous recombination

pA
ITR ϕ ITR
p cDNA
cDNA pA
rAd-cDNA [E1 and E3 (partial) deleted]
PNAS 95:2509 1998
(He, .., Vogelstein)
Replication competent Ad (RCA)
contaminant
• Recombination of rAd with the E1 region
integrated in 293 cell.

pA
ITR ϕ ITR
p cDNA
cDNA pA
rAd-cDNA [E1 and E3 (partial) deleted]

Wild type adenovirus

 Late genes
pTP ψ ITR

POL ITR ∆E1 E2 ∆E3 E4

ssDBP

E1-like activity from infected cell

Late gene
Immune response
expression

Clear by immune system

within 2 to 6 weeks
 Second-Generation Ad
• Remove E2a, E2b, or one of the ORF of E4
plus the E1/E3
• Less immune response
• Prolonged expression period, several
months (more than 6 months vs. 2 to 6
weeks of the first-generation Ad)
 Late genes
pTP ψ ITR

POL ITR ∆E1 E2 E3 E4

ssDBP

E1-like activity?
延長基因表現時間
降低免疫反應

Late gene
Immune response
expression
 Helper dependent Ad
(3rd generation Ad)
• Gutless Ad or Gutted Ad
• Recombinant Ad without viral gene
• Only cis elements included,
– ITR and packaging signal
• Helper Ad needed to produce HD virus
• Helper Ad contamination (0.01%: 108 – 1012)
• Problems with large-scale production
 Proc. Natl. Acad. Sci. USA
Cre/loxP: bacteriophage P1 Vol. 93, pp. 13565–13570, 1996
 Adenovirus ∆E1b-55kD cancer gene therapy

ψ Late genes ITR

pTP
POL ITR
ssDBP ∆E1b E2 E3 E4

E1
Specific promoter: only replicate in targeted cells
Infect cells
Kill cells

Late gene Packaging

expression

Replicates in p53- cells only

 CV890, AFP promoter
• With α-Fetoprotein (AFP) transcriptional
regulatory elements (TRE)
• AFP, a tumor marker currently used for the
diagnosis and management of HCC
• HCC-specific oncolytic adenoviruses
• TRE drive bicistronic E1A-IRES-E1B cassette
• Combination with doxorubicin
• Significant reduction of tumor size
• Cancer Res. 2001 Sep 1;61(17):6428-36.
Biosynthetic pathway of PGI2
Phospholipid Arachidonic Acid (AA)
Phospholipases A2

COX
PGH2 (PGG2)
PGIS
TXAS

PGI2 TXA2 PGE2, PGD2, PGF2α

6-keto-PGF1α
ECV304 cells
50 50 50 50 50 0 10 20 100 Ad-COX-1 (MOI)
0 10 20 50 100 50 50 50 50 Ad-PGIS (MOI)
COX-1

PGIS

HUVEC
MV
Ad r o l
-C

50 50 50 50 50 0 20 100 Ad-COX-1 (MOI)

nt
Co

0 10 20 50 100 50 50 50 Ad-PGIS (ΜΟΙ)

COX-1

PGIS
PGs profile of ECV304 cells infected with Ad-COX-1 and/or Ad-PGIS

COX-1 (50) PGIS(50)

AA AA
PGE2
PGF2α HHT

6-KP COX-1 (50)/PGIS(10) PGIS(50)/COX-1 (10)

AA
AA
HHT
6-KP

6-KP COX-1 (50)/PGIS(20) PGIS(50)/COX-1 (20)

AA AA
6-KP
HHT
HHT
COX-1 (50)/PGIS(50) PGIS(50)/COX-1 (50)
6-KP 6-KP

AA AA

HHT HHT

COX-1 (50)/PGIS(100) 6-KP PGIS(50)/COX-1 (100)

6-KP
AA
AA
HHT HHT

Circulation 2001;103:2090-2095
• First generation Ad
– E1 plus E3 deletion: 5 ~ 7 kb
– Stronger immune response of E3 deleted Ad
– Leakage of some Ad peptides expression which may cause host immune
response
• Second generation Ad
– Part of E2 and/or E4 deleted
– Complementary cell line needed
– Much less Ad peptides expressed, prolonged gene expression; several
months
• Helper dependent Ad (Gutless or Gutted)
– Containing only cis elements, ITR and Ψ, needs helper virus
– Very large capacity, up to 35 kb
– Very low immunogenesity, different serotypes can be used for repeat
admissions,
– No Ad gene expressed, at least a year of high expression level, up to two
years in mice.
 Gene Therapy (1999) 6, 1565:573

Use of helper-dependent adenoviral

vectors of alternative serotypes permits
repeat vector administration
RJ Parks 1,2,3 , CM Evelegh 1 and FL Graham 1,2
Departments of 1 Biology and 2 Pathology, McMaster University, 1280 Main Streeet West,
Hamilton, Ontario, L8S 4K1, Canada
 Ad Gene Therapy on Trial
• 17 September 1999: Dr. J. Wilson, Univ. Penn. in
Phil. – Jesse Gelsinger (OTC deficiency) died with
38 trillion Ad particles, directly liver injection
• E1 and E4 deleted Ad
• Died from a massive immune response to Ad vector
• High and sustained levels of interleukin-6;
• Dose related IL-6 increase 2 to 4 hours after
injection
• Precursor of red blood cells had been wiped out:
maybe due to a preexisting parvovirus infection
• Four days later he died of multiple organ failure
 Ad Gene Therapy on Trial
• Very low CAR in human liver compared to mouse liver,
misleading of rodent model?
• Much higher dose needed for human liver gene transfer,
low gene transfer rate (<1%)
• 25 cancer patients (R. Warren) with as high dose with
no such problems
• Empty capsids appear to be immunogenic like intact
virus
• Patient was very sick and not qualified for gene therapy
 Compliment Activation by Adenovirus

• Complement system: a natural and powerful

defense against invading pathogens
• Activation only in samples that contained
antibodies against adenovirus
• Cause inflammation in multiple tissue:
failure
• Screen for their complement response
before Ad gene therapy (Other sources?)
• Gene Therapy 8, 1794 - 1800 (09 Jan 2002)
 Adeno-Associated Virus
• ~ 4.7 kb, single strand DNA
• 80-90% of adults are seropositive
• Not associated with either symptoms or disease
• Site-specific integration into genome at 19q13.3-qter;
mediated by Rep 68/78 proteins
• Infect vertebrates and invertebrates
• 20-30 nm non-enveloped, may pass through gap
junction
• Helper virus required; adenovirus or herpes simplex
virus (HSV)
Genomic map of AAV
Recombinant AAV construction
Production of recombinant AAV
particles
 AAV production and purification
• 3 plasmid transfection system: up to 1x1010 viral
titer/10 cm plate
• Packaging cell line established
– Rep and Cap are toxic: regulated by tetracycline-
inducible system
• Purification:
– Precipitate by ammonium sulfate followed by CsCl
or iodixanol centrifugation
– Ligand affinity chromatography: heparin columns
– 50%- 70 % recovery with >99% purity and higher
particle-to-infectivity ratios (100:1)
 Problems of AAV
• Small inert size, limited to 5 kb
• Labor-intensive production, rolling bottles,
still have problem
• Random integration: One study showed mice
with tumor, ~2 years after rAAV injections
• Immune response limited the 2nd injection,
same as all viral vectors and liposome
 AAV for larger size insert

• Split transgene into two

separate vectors
• Both vector infect target
cell: form episomal
circular multimers in
head-to-tail orientation
• RNA spicing needed
• 16% to 70%: conflicting
results
 AAV integration site
• Miller et al., Nat. Genet. 30:147; 2002
• J Virol. 2005, 79:11434
• rAAV with neomycin gene
• Hela cells
• Deletion of AAV genome
• Deletion of cell genomic sequences
• Unknown sequences insertion
Localized AAV integration sites vs.
random sites

Miller et al., J Virol. 2005, 79:11434

Distribution of chromosomal integration
sites of AAV

Miller et al., J Virol. 2005, 79:11434

Miller et al., J Virol. 79:11434, 2005
@ Efficiently package dsAAV after mutating one ITR.
 AAVs infect muscle in vivo
1W (30’) 2W (2’) 6W (2’) 6M (2’) 6W

dsAAV

ssAAV

1W (30’) 2W (30’) 6W (30’) 6M (30’) 6W

Treatment: I.M. (tibialis interior), 1x1011 v.p./50 µL

AAV: An Amazing Vector

 Double Strand AAV
1. A new strategy for efficient AAV production and accelerated
transgene expression in vitro and in vivo.
2. Overcome barriers in poor permissive cells.

3. Long-term and increased gene expression in vivo.

4. In hepatocytes, dsAAV forms supercoil structure.

5. Decreased size of foreign genes could be packaged (2.5K).

 A Phase I/II Clinical Trial for Liver
Directed AAV-Mediated Gene Transfer for
Severe Hemophilia B.
• Factor IX
• Dose up to 8 x 1012 vg/kg to skeleton muscle
(safety study)
• Liver-directed gene transfer has a 10-50 fold
dose advantage and reduced probability of
forming inhibitors when compared to muscle
delivery
• Life-long correction of the bleeding diathesis in
hemophilia B mice (Liver)
• Up to 14% of wildtype levels of canine F.IX in
hemophilic dogs
• Hemophilia B dogs followed for >4 years still
maintains the initial level of F.IX following the
administration of AAV-2
• 1 x 10e12, 2 x 10e12 vg/kg (human; liver)
• Blood chemistries including the liver enzymes and
blood counts remained normal after treatment
• Small amounts of vector DNA were transiently
detected in body fluids including the semen
• 6-8 weeks is required to reach plateau levels of
transgene expression
• AAV-F.IX to humans at doses up to 2 x 10e12
vg/kg was well-tolerated with no acute toxicity
• LST (liver index) up-high after 4 weeks of injection?
• Clinical trial stopped, 2004, due to liver
toxicity.
Retrovirus (MLV)
 Retrovirus
• RNA virus
• Infect primary cells
• 5’ LTR as promoter, 3’ LTR as terminator
• LTR (U3-R-U5): promoter, poly A
– U3: transcription starting site; att (5’) site for integration
– U5: att site (3’),
• Viruses are budded and released to culture medium:
collected for purification
 Retrovirus transduction system
• Integration into chromosome: provirus
– Stable transduction
– High efficiency
– Non-specific integration
– Requires mitosis: dividing cells only
• Replication-incompetent vectors
• Cis elements:
– LTR, RNA primer binding site and polypuring tract (PPT)
– Packaging signal: Ψ: for encapsulation
• Replacement of U3 in the 5’ LTR: 100-fold increase
in viral titer and high transcriptional activity
 Retrovirus transduction system
• Minimize recombination to produce virus: split
genome to two parts
• VSV-G: G protein of vesicular stomatitis virus:
pseudotyped viruses: replace env protein
– Extreme broad host range of VSV (Bind nonspecifically to
phospholipid)
– More stable: purified to higher titer (1000 x)
• Titer: > 109 IU/ml ; (~106 without VSVG)
• High infection efficiency needed to target stem cells.
• Commercial available (Clontech and …)
Replication-incompetent（無法自我複製）retrovirus

gag and pol

expressing cell

Co-transfect with
pVSV-G
*no poly A signal at 3’ end: replacement of U3/R
*mutation in the U3 region of 3’ LTR: increase biosafety
 Lentiviral vector
• Complex retrovirus (lente: slow)
• Infect dividing and non-dividing cells
(MLV retrovirus can only infect dividing cells)
• Nuclear targeting without nuclear
membrane disassembly
• Integrated into host genome
• Vpr is dispensable, lack in lentiviral
constructs
 Lentiviral vector modification
• Replacement of HIV envelop glycoprotein with
VSV-G: first useful lentiviral vector
• Viral particle can be concentrated (~1000-fold) by
ultracentrifugation
• Titers: 1 x 109 – 1 x 1010 IU/ml
• 293T packaging cells
• Production by cotransfection of several
recombinant lentiviral plasmids
• Other lentivirus packaging systems: Feline IV (貓),
Simian IV (猴), HIV-2, Equine IVA (馬), and
maedi/visna virus (sheep disease; maedi which effects the lungs; visna
which effects the central nervous system)
 J. Gene Med. 2:308;2000

• Packaging cell: gag, tat and VSV-G are toxic need

to use tetracycline-regulated system.
• SIN and 3rd-generation lentiviral packaging
system available. (Invitrogene)
 Stable transduction of quiescent
DC34+CD38- human hematopietic cells
by HIV-1-based lentiviral vector
• Compared with MLV
• GFP as reporter
• Non-dividing DC34+ cells: 45.5%
• DC34+CD38- cells in G0: 12.4%
• Stable transfection of DC34+CD38- cells for over 15 weeks
• Both MLV and lentiviral vectors efficiently transduced
cytokine-stimulated CD34+ cells

PNAS 96:2988, 1999.

Hybrid vectors

Nat. Biotechinol. 15, 886-70 (1997)

 Gene Therapy of Human SCID-X1
Disease
• Science 288:669 (2000); A. Fisher, Paris, France
• Mutation at γc cytokine receptor subunit
• 1st success case (two patients) of gene therapy
• CD34+ cells; Retrovirus;
• 19 and 17 x 10E6 CD34+ cell were infused
• ~ 20 to 40% of cells expressed γc transgene
• Leave protection at 3 months
• 10 months after transducing: normal in
numbers of T, B, and NK cells: Full correction
Gene Therapy of Human SCID-X1 Disease
 SCID-X1 gene therapy

• 11 more patients
• 30 months later one patient develop
leukemia
• Inserted into more than 40 sites in the
genome of different repopulating cells
• Abnormal T-cell: inserted in the Lmo-2
oncogene
• Nature Medicine 8:1189 (November 2002)
 SCID-X1 gene therapy
• March 15 2003; two of the 11 boys treated
developed leukemia
• May insert near Lmo2 in 1/100,000
• Millions of bone marrow cells are modified and
returned
• Third child with an insertion near Lmo2 gene;
developed leukemia later.
• Many projects on hold
• London UK: it would treat X-SCID if “death was
otherwise inevitable”
Figure 1. A Potential Mechanism
of Leukemogenesis.
T-cell leukemia developed in two
children with SCID who were
treated with CD34+ stem cells
transduced with a viral vector
containing IL2RG, which encodes
the common γ subunit of the
interleukin-2 receptor. A recent
study by Davé et al. suggests that
the γc transgene was inserted close
to the LMO2 gene, activating the
T-cell oncogene. Overexpression
of the LMO2 protein in T cells
blocks the differentiation of the
cells and thus increases their
susceptibility to leukemia. This
finding has potential implications
for the design of gene-therapy
protocols.
n engl j med 350;16, April 15, 2004
 Good News for Gene therapy
• The γc gene can act as an oncogene when
under control of the retroviral promoter
• Both γc and Lmo2 act as oncogenes;
collaborating oncogenes: double hits
• In most gene therapy applications, the
therapeutic gene does not have oncogenic
potential.
• Improving gene therapy: not to activate
juxtaposed genes
Gene therapy for inherited genetic
defects

Nat Biotechnol. 2006 Aug;24(8):949-50

Targeted gene correction by AAV

Nat Biotechnol. 2006 Aug;24(8):1022-6

Nat Biotechnol. 2006 Aug;24(8):949-50
 Clinical trials for gene therapy
• Over 400 clinical trials have been conducted or are
underway
• More than 6000 patients
• > 70% are cancer related
• Immuno-modulation, tumor associated antigen, tumor
vaccine
• Suicide genes and prodrugs
• Tumor suppressor genes
• Anti-angiogenesis genes
• Conditional replicative adenovirus
Human gene transfer clinical trials
• http://www4.od.nih.gov/oba/rac/clinicaltrial.htm
• http://www.wiley.co.uk/wileychi/genmed/clinical/
 Enzymes for cancer gene therapy
• Bystander-killing effect:
– A percentage of the tumor cells needs to be
transduced for eradication of a tumor
– Can not treat secondary, distal tumors that are not
directly accessible by gene transfer
 GCV/TK
Tumor cells can be killed by:
• Direct effect on the transduced tumor cells
• Bystander effect through cell gap junctions and
kill neighboring cells
• Local inflammatory effect caused by the injected
mouse cells
• Systemic immune response
• Mutated HSV-tk to generate more sensitive tk
– Random oligonucleotide mutagenesis
Nanomolar IC50 (30 µM for WT)
– 10 fold lower dose of GCV
– 35 fold increase in the Km for thymidine
– Liver injury causes by systemic delivery of GCV
Immunomodulatory gene therapy
• Cytokines, co-stimulatory molecules,
tumor-associated antigens and/or major
histocompatibility complex Class I
molecules have been delivered to tumors
• Immunostimulatory Cytokines: activation of
tumor-specific T lymphocytes; rejecting
tumor cells, protecting from recurrence
 Anti-angiogenesis therapy
• Endostatin (angiostatin and TIMP-2)
– Ad-endostatin: increase plasma endostatin to 605-1770
ng/ml (was 8-33 ng/ml)
– Systemic administration
– Reduce tumor volume
– Combination of radiotherapy and Ad-angiostatin
– Protease gene transfer to generate kringle 1-3 segments
– Using AAV as vector
– Cancer-Res. 2000 Mar 15; 60(6): 1503-6
– PNAS. 2000 Jun 6; 97(12): 6698-703
– PNAS. 2000 Apr 25; 97(9): 4802-7
 VEGF inhibition
• Over express extracellular domain of VEGF
receptor in a remote organ
– Ad-VEGF-ExR: flt-1 fused to the Fc portion of human
IgG.
– Soluble receptor secreted from Ad-VEGF-ExR infected
cells bound to VEGF and inhibited VEGF induction in
EC.
– Injected into skeletal muscle
– Receptor can be detected in blood for 3 weeks, tumor
growth ceased after 10 days and size declined thereafter.
EPO
 The Future viral vectors
• Can be injected
• Target to specific cells
• Safe and efficient gene transfer: high
percentage of transfection
• Insert themselves into appropriate regions of
the genome ( or persist as stable episomes)
• Regulated either by administered agents or
by the body’s own physiological signals.
• Cost-effective to manufacture
 The future viral vectors for
gene therapy
• Large-scale production
• Cure disease
• Very high efficiency of gene transfer and a
large number of gene-therapy vectors:
billions of cells
• Site-specific integration, in situ homologous
recombination, …. Takes much longer
• Our imagination is the limit…..
 Cardiovascular gene therapy

• Angiogenesis gene therapy

– VEGF, FGF-1, FGF-2,
– Endothelial progenitor cells (EPCs)
– Bone marrow derived EPCs
– Stimulate hematopoietic progenitor cells with GM-
CSF
DNA targeting by AAV is enhanced
by DNA double-strand breaks

Miller et al., MCB 23:3550, 2003

 Liposome protects Ad from
neutralising Ab

Dilution of serum 1 to 128 Infection efficiency

Journal of Virological Methods 126 (2005) 31–36

Targeting adenoviral vectors using sCAR
bifunctional fusion protein
 Stable transduction of large DNA by
high-capacity adeno-associated
virus/adenovirus hybrid vectors
• Using Ad to carry AAV
• Stable integration of AAV in cells
• Larger capacity in AAV due to Ad

J. Virol. 2002; 76, 10734– 10744

 Ad-AAV hybrid vectors
Stable transduction of large DNA by high-capacity adeno-
associated virus/adenovirus hybrid vectors
Viral invasion of infected cell nucleus

Cullen BR. Cell, Vol. 105, 697–700, 2001