viruses as vectors any virus can potentially be used to express foreign genes different viruses are...
TRANSCRIPT
Viruses as Vectors
Any virus can potentially be used to express foreign genes
Different viruses are better suited for different kinds of uses
Integration may be important, such as in many gene therapy uses
Larger viruses can express more and larger foreign genes but are more difficult to manipulate
The cis-acting promoters for genome replication and packaging must be understood
Potential Uses of Viral Vectors
Gene therapy to replace a missing or inadequate gene
Cure of illness by expressing a reagent to, for example, kill cancer cells
Immunization by expressing an antigen from a pathogen
Expression of genes in cultured cells for scientific study
Features Retroviral Lentiviral
Vectors Based on RNA Viruses
Alphaviral
Maximum Insert size 7-7.5 kb 7-7.5 kb 5 kb
Concentrations viral particles/ml
>108 >108 >109
Route of gene delivery
Ex vivo Ex/In vivo In vivo
Integration Yes Yes No
Duration of expression in vivo
Shorter than theorized
Long Short
Stability Good Not tested Good
Ease of Preparation scale up
Pilot scale up up to 20-50 liters
Not known Not known
Immunological problems
Few Few Unknown
Pre-existing host immunity
Unlikely Unlikely, except in AIDS patients
No
Safety problems Insertional mutagenesis?
Insertional mutagenesis?
Few
Adenoviruses Adeno- associated viruses
Vaccinia virus
Vectors Based on DNA and on DNA Viruses
Naked DNA /Lipid DNA
HerpesvirusesFeatures
No Yes/No No very poorNoIntegration
Good Good Good Very goodUnknownStability
Extensive Not known Extensive NoneNot knownImmunological problems
Yes Yes Diminishing as unvaccinated population grows
NoYesPre-existing host immunity
7.5 kb 4.5kb >25 kb Unlimited size~30kbMaximum Insert size
>1010 >1012 107-109 No limitation>108Concentrations viral particles/ml
Ex/In vivo Ex/In vivo Ex/In vivo Ex/In vivoEx vivoRoute of gene delivery
Short Long Short ShortShort/ Long in CNS?
Duration of expression in vivo
Disseminated vaccinia in immunocompromised hosts
Inflammatory response, toxicity
Inflammatory response, toxicity
None?Neurovirulence? Insertional mutagenesis
Safety
Easy to scale upDifficult to purify, difficult to scale up
Vaccine production facilities exist
Easy to scale upNot yet triedEase of Preparation scale up
FG
Plasmid
TK
wt vaccinia virus
Homologous recombination
TK+
BUdR selection
Virus production
TK-
Recombinant vaccinia virus
Nucleus
FG
FG
FG
TK Gene flanking sequences and foreign gene (FG)
Bacterial
Generation of a Recombinant Vaccinia Virus Expression Vector
E1AE1B
E2A
E3
E4
L1 L2 L3 L4 L5
E2B
RL
Virion Structural ProteinsA. Wild type Adenovirus Genome
E1
Complementing Cell Line
B. Adenovirus vector DNA (E1, E3 deleted, expression cassette inserted)
Expression cassette
Endosome
Product of expression cassette
Transfect adenovirus vector DNA into complementing cell line that expresses the E1A gene
DNA
Target Cell
Vector DNA packaged into virion particles
Infect target cellEpichromosomal action
Generation of a Non-replicating Adenovirus Expression Vector
Mammalian type C Retrovirus DNA lacking
Packaging Cell Line
IntegrationA
B
RT
CapsidGlycoproteins
LTRLTR gag pol env
RT
Expression cassetteRNA
DNARandom integration
Expression Products
E
F
G
Reverse Transcription
vector
proviral
D
Expression cassette vector
Retrovirus vector DNAExpression cassette
wt RNA
C
Glycoproteins
RTVector RNA wt RNA
Producer Cell Line
Transfect packaging cell line
Integration
Capsid
Expression cassette
Infect target cell with packaged vector
Target Cell
Generation of a Packaged Replication-deficient Retrovirus Expression Vector
Replicase proteins Structural proteins
SG
CAP A n
A. Alphavirus genome organization
Replication promoters
Subgenomic promoter
Encapsidation signalForeign gene
SG
B. Alphavirus replicon packaged with a nonpackaged helper.
StructuralReplicase
CAP
CAP An
An
SG
SG
Structural proteins
ReplicaseCAP
C. Nondefective alphavirus expression vectors with two subgenomic promoters
ReplicaseCAP
An
An
SG SG
SG SG
Structural proteins
Structural proteins
Alphavirus Expression Vector
P1 P2 P3
2 3 14
A) Poliovirus infectious clone
3B3A 3C 3D2C2B2A
5’NTR
T7EcoRI
Poly (A)(60nt)
B) Poliovirus replicon encoding TNF-
P1 P2 P3
243B
3A 3C 3D2C2B2A5’NTR
T7EcoRI
Poly (A)(60nt)
TNF-
2Apro
T-Y G-V-D-L-R V-N-T-K-D-L-T-T- Y G
2Apro
SalI
SalI
Poliovirus Replicons for Expression of Foreign Genes
VSV vcDNA
le N P M G L tr
5’3’
vRNA (-)
vcDNAT7
A.
B.
T7
pL
pN
pP
T7
T7
T7
Coinfection and cotransfection of the Necessary Components of the System
Vaccinia expressing T7 polymerase
Plasmids with T7 promoters encoding VSV P, N, and L
Rhabdovirus (VSV) genome organization
T7
Expressing a Negative-Strand RNA virus from cloned cDNA(part 1)
(part 2)Expressing a Negative-Strand RNA virus from cloned cDNA
Glycoproteins
Infectious VSV
T7 RNA polymerase
VSV G protein
VSV L protein
VSV N, P proteins
M protein
An An AnmRNAs
N,P proteins
vRNP
L
An
M protein
T7 RNA polymerase
(L, P, N proteins)
vcRNP (+)
C. Expression of VSV replicase proteins (L, P,) and N protein under the control of T7 promoter
D. Replication of VSV vRNA from cDNA by T7 polymerase; replication of vcRNA, vRNA, and transcription of VSV mRNAs by VSV replicase/transcriptase
E. Translation of viral proteins and assembly of infectious virus.
A. Genome organization of mutant VSV plasmid
Mutant vcDNA
le N P M L tr
5’3’
T7
T7
pL
pN
pP
T7
T7T7
Vaccinia expressing T7 Polymerase
Plasmids with T7 promoters encoding VSV P, N, and L
CD4 CXCR4Mutant vRNA (-)
Mutant vcDNA
T7
Expression of a Pseudotypic VSV with CD4 and CXCR4 in place of the VSV G Protein(part 1)
B. Coinfection and cotransfection of the Necessary Components of the System
C. Expression of VSV replicase proteins (L, P,) and N protein under the control of T7 promoter
D. Replication of VSV vRNA from cDNA by T7 polymerase; replication of vcRNA, vRNA, and transcription of VSV mRNAs by VSV replicase/transcriptase
Expression of a Pseudotypic VSV with CD4 and CXCR4 in place of the VSV G Protein(part 2)
E. Translation of VSV M, N, P, and L and CD4 and CXCR4
vRNP
T7 RNA polymerase
VSV L Protein
VSV N, P Proteins
CD4 Protein
CXCR4 Protein
Expressed Products:
M protein
T7 RNA polymerase
(L, P, N proteins)
vcRNP (+)
CD4
An An
N,P proteins
mRNAs
L
AnAn
M proteinCXCR4
An
F. Assembly of Pseudotypic VSV
F. Infection of Healthy and HIV-infected cells by Pseudotypic VSV
Expression of a Pseudotypic VSV with CD4 and CXCR4 in place of the VSV G Protein(part 3)
HIV-infected cell
Becomes infected with
mutant VSV
HIV-infected cell dies
Eukaryotic Cell Susceptible to VSV
Not infectible with
mutant VSV
Infectious VSVWith CD4,CXCR4
(mutant VSV)
Healthy cell lives
C prM E NS1 NS5
Infectious 17D yellow fever cDNA clone
A. Original constructs
JE (Nakayama) cDNA (structural proteins of virulent strain)
JE (SA) cDNA (structural proteins of human vaccine strain)
B. Chimeric constructs
C prM E NS1 NS5
YF/JE-S prM-E
Titer after RNA transfection of
VERO cells
PRNTanti-YF Anti-JE
6.3 x 106
YF/JE-N CprM-E
<10 NA NA
<1.3 3.1
YF/JE-N prM-E
2.0 x 107 <1.3 3.4
YF/JE-S C prM-E
<10 NA NA
Yellow fever (17D vaccine strain) chimeras with JE proteins for use as a JE Vaccine
Genetic Defects that are Candidates for Gene Therapy
Disease Defect Incidence Target Cells
Severe combined immunodeficiency (SCID)
Adenosine deaminase (ADA) in 25% of SCID patients
Rare Bone-marrow cells or T lymphocytes
Familial hypercholesterolemia
Deficiency of low-density lipoprotein (LDL) raeceptor
1:1 million Liver
Cystic fibrosis Faulty transport of salt in lung epithelium
1:3000 Caucasians Airways in the lungs
Hemoglobinopathies thalassemias
(Structural) defects in the or globin gene
1:600 in certain ethnic groups
Gaucher’s disease Defect in the enzyme glucocerebrosidase
1:450 in Ashkenazi Jews
Bone marrow cells, macrophages
1 antitrypsin deficiency
inherited emphysema
Lack of1 antitrypsin 1:3500 Lung or liver cells
HemophiliaB Factor IX deficiency 1:30,000 males
A Factor VII deficiency 1:10,000 males Liver, muscle, fibroblasts or bone marrow cells
Duchenne muscular distrophy
Lack of dystrophin 1:3000 males Muscle cells
Cancer Many causes, including genetic and environmental
1 million/year in USA Variety of cancer cell types, in liver, brain, pancreas, breast, kidney
Neurological diseases Parkinson’s, Alzheimers’s spinal-cord injury
1 million Parkinson’s and 4 million Alzheimer’s patients in the USA
Neurons, glial cells, Schwann cells
Cardiovascular Restenosis, arteriosclerosis
13 million in USA Arteries, vascular endothelia walls
Infectious diseases AIDS, hepatitis B Increasing numbers T cells, liver, macrophages
Rheumatoid arthritis Autoimmune inflammation of joints
Increasing numbers with aging population
Some Acquired Diseases that are Candidates for Gene Therapy
Disease Defect Incidence Target Cells
Clinical Trials of Gene Therapy for Monogenic Diseases in the United States in 2000
Disease Gene Number of patients
ResultsVector Number of Trials
One trial shows long term elevation of GC expression, other trials primarily Phase I
Gaucher disease 9GCc Retrovirus 3
OTC deficiency OTC Adenovirus 1 14 Trial suspended after one fatality (see text)
ADA-SCID ADA + NeoR Retrovirus 1 6 Ongoing since 1990
Cystic Fibrosis Adenovirus Some correction of defect in 30% of patients, but inflammation at clinical doses, and reduction in therapeutics with repeated injection.
CFTR 9 83
AAV Some correction of defect, Phase II study started
36CFTR 4
Cationic Lipids
25 30% to 50% of patients showed showed improvement
CFTR 4
Chronic granulomatous
p47 phox/ gp91 phox
Retrovirus 3 9 Phase I/II, study closed in 1998
Familial hypercholesterolemia
LDLR Retrovirus 1 5 Phase I, closed in 1994
Rheumatoid arthritis RetrovirusIRAP 1 7 ?
Artery disease and restenosis
VEGF Naked DNA 2 29 ?
Chronic Diseases
Infectious Diseases
AIDS HIV-IT(V) Retrovirus 3 298
CD4-Zeta TcR Retrovirus 3 54
Anti-HIV ribozymeRetrovirus 2 12
TK + HyR Retrovirus 2 14
Antisense to TAR Retrovirus 3 17
Most gene trials for HIV are in Phase I, with a few in phase II. Few results reported.
Other Clinical Trials of Gene Therapy in the United States in 2000
Disease Gene Number of patients
ResultsVector Number of Trials
Clinical Trials of Gene Transfers for Cancer Therapy in the United States in 2000
Location Gene Number of patients
PhaseVector Number of Trials
Brain cancersNeuroblastoma IFN Retrovirus 1 4 I
IL-2 Retrovirus 1 12 IIL-2 Adenovirus 1 6 I
Central nervous systemTK Adenovirus 2 22 IPediatric tumor TK Retroviral producing cells1 2 IAdult brain tumor TK Retroviral producing cells1 15 I
Ovarian cancer HSV-TK Adenovirus 1 10 ITK Retroviral producing cells3 42 IBRCA-1 Retrovirus 1 40 I/IIp53 Adenovirus 1 16 I
Small cell lung cancer IL-2 + NeoR Lipofection 1 8 IAnti-sense to k-ras
Retrovirus 1 9 I
p53 Adenovirus 2 59 I/II
Prostate cancer GM-CSF Retrovirus 1 8 I/IIPSA Poxvirus 1 3 IHSV-TK Adenovirus 1 18 I
E1A Lipofection 1 16 IBreast cancer BRCA-1 Retrovirus 1 21 I
MDR-1+ NeoRRetrovirus 4 39 ICD80 Lipofection 1 15 ICEA Poxvirus 4 53 ICEA RNA transfer 1 30 I
Melanoma GM-CSF Gene gun 1 17 IGM-CSF Retrovirus 2 29 IHLA-B7/b2m Lipofection 8 165 I/IIIL-2 + NeoR Retrovirus 5 115 I
1FN Retrovirus 3 91 ITNF+NeoR Retrovirus 1 12 I/IIMART-1 Adenovirus 1 33 IMART-1 Poxvirus 2 16 Igp100 Poxvirus 1 19 Igp100 Adenovirus 1 7 ICD80 Lipofection 1 17 I
Miscellaneous carcinomas
p53 Adenovirus 1 26 IHLA-B7/b2m Lipofection 4 76 IIIL-2 Lipofection 1 11 ICEA Poxvirus 1 8 I
Lymphomas and solid tumors
IL-2 Retrovirus 2 29 IRetrovirusTK 1 11 IRetrovirusIL-12 + NeoR 1 31 I
AdenovirusBladder cancer p53 1 5 I
Colo/rectal, renal, and liver cancers
CD Adenovirus 1 6 I
TNF+NeoR Retrovirus 1 12 I
GM-CSF Retrovirus 1 18 IHLA-B7/b2m Lipofection 4 53 I/II
IL-4 Retrovirus 1 18 I
Clinical Trials of Gene Transfers for Cancer Therapy in the United States in 2000
Location Gene Number of patients
PhaseVector Number of Trials
(continued)
Gene Therapy--An Apparent Success
Restenosis--reblockage of coronary arteries after they have been opened by coronary bypass surgery or angioplasty
13 patients with restenosis were injected in the heart with DNA encoding vascular endothelial growth factor, which promotes angiogenesis
All 13 patients had improved heart function
Gene Therapy--A Partial Success
SCID--Severe Combined ImmunoDefficiency--is often due to the failure to produce adenosine deaminase (ADA)
The accumulation of adenosine is particularly toxic for T cells and both humoral antibody and CTL functions are abolished
Treatments include bone marrow transplant (BMT) if a suitable donor can be found or injection of ADA 1-2 times/week
10 people have been treated with T cells that were infected ex vivo with retroviral vectors expressing ADA
Most now express ADA but not enough to do away with the ADA injections
Gene Therapy--Another Apparent Success
SCID XI, caused by lack of the c subunit of the receptors for IL-2, -4, -7, -9, and -15, can be treated only by isolation in a sterile bubble followed by BMT, if a donor is available
Stem cells from bone marrow from three infants with SCID XI were infected ex vivo with retrovirus expressing the missing gene and the cells reinfused into the donors
The 3 infants produce T, B, and NK cells and are apparently healthy
Long term followup will be required to determine if the cure is permanent
They have been successfully received a number of childhood vaccinations
Gene Therapy--A Failure
Jesse Gelsinger, a young volunteer in a gene therapy trial who had a moderate OTC defficiency, died on 17 Sept 1999
He had been injected in the liver with high concentrations of adenovirus that expressed OTC
He apparently died of a massive immune response to the adenovirus vector
Inability to produce ornithine transcarbamylase (OTC) is often lethal, but moderate deficiencies may be controlled by strict control of diet