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