GENE THERAPY: To correct a genetic defect by transferring of a

functional normal copy of the gene into cells

Examples of diseases caused by genetic defect Ornithine transcarbamylase (OTC deficiency) Hemophilia (blood coagulation factors VIII or

IX) SCID( severe combined immunodeficiency) Muscular dystrophy Cystic fibrosis Sickle cell anemia

Viral Vectors: Adenovirus Retrovirus Lentivirus Adeno-associated virus (AAV) Herpes simplex virus (HSV)

Non-viral vector based Naked DNA (plasmid DNA): injection or genegun Liposomes (cationic lipids): mix with genes

Ex-vivo In vivo

Virus are obligate intracellular parasites

Very efficient at transferring viral DNA into host cells

Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins)

Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted

Replication-competent virus Replication-defective virus

Amplicon: doesn’t encode structural proteins

Can’t replicate beyond the first cycle of infection

Elements needed to generate amplicon Transfer Vector: plasmid (promoter, gene

of interest, ori, packaging signal) Packaging vector (cosmid or cell lines):

provide the viral structural proteins for packaging of transfer vector

Helper virus (packaging of transfer vector): deleted Packaging signal sequence

Amplicon Vector System

Cotransfection with HSV-1 bacmid lacking packaging sequences ("helper genome")

Amplicon Plasmid

oripac

Plasmid is replicated into concatemers, cut into genome-unit length molecules & packaged into viral particles by the helper genome; the helper

itself cannot be packagedHelper-free amplicons

Non-enveloped ds DNA, 36 kilobases Early proteins (E1A, E1B, E2,E3 and

E4), late proteins (L1-L5) Causes a benign respiratory infections

in human Serotypes 2 and 5 are commonly used

as vectors

Adenovirus fiber binds to CAR (coxsakie and adenovirus receptor, CAR), receptor which is ubiquitous

Modify the fiber protein

Advantages High titers Both dividing and non-dividing cells Wide tissue tropism Easily modify tissue tropism

Disadvantages Transient expression ( not good for genetic

diseases) Highly immunogenic High titers of virus can be toxic More suitable for cancer immunotherapy

Moloney murine leukemia virus (MuLV) Generation of replication defective retroviral

vector Transfer plasmid vector:

Gene of interest Long terminal repeats(LTR): promoter, polyA,

integration, replication and reverse transcription Primer binding site (PBS) (origin of replication) RNA packaging signal Poly purine tract (important for replication)

Packaging vector Cell line stably transfected with plasmid

constructs containing Gag/pol and Env.

Advantages Integration: permanent expression Pseudotyped virus

Disadvantages Only infecting dividing cells Insertional mutagenesis (tumor formation)

Activate oncogenes Inhibit tumor suppressor genes

Infection of non-dividing cells (hepatocytes, neurons)

HIV, a human lethal pathogen Delete accessory

genes Provide an envelope

from a non-retrovirus (VSV)

Develop vectors from lentiviruses of non-human pathogens SIV, FIV, EIAV etc

Herpes simplex virus 1, mild disease in human, no risk

Linear ds DNA, 152 kb, about half of the total 81 genes are non-essential for virus replication

40-50 kb of foreign DNA can be accommodated

Neurotropic virus, target to nervous system

Replication defective amplicon particles

Non-pathogenic human parvovirus, non-enveloped ss DNA virus, 4.6 kilobases

Dependent on a helper virus ( adenovirus or herpesvirus) for replication (dependovirus)

AAV-2 mostly used for vector

Advantages Integration and persistent expression No insertional mutagenesis Infecting dividing and nondividing cells Safe

Disadvantages Size limitation, 4.9 kb Low titer of virus, low level of gene expression

Viral vector titers manupilation of immunogenicity infecting of

tropism non-dividing cells

Adenovirus 1011 terrific very high yes

Retrovirus 107 good low only lentivirus

Herpesvirus 107 not so good low yes

AAV 107 not so good low yes