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