Gene Transfer

Comparison of different viral vectors

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

2nd generation lentiviral plasmids

3rd generation lentiviral plasmids

Retroviral vector• 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

Generation of retroviral vector

Pseudotyped retroviral vector

Characteristics of retroviral vector

• Advantages– Integration: permanent expression– Pseudotyped virus

• Disadvantages– Only infecting dividing cells– Insertional mutagenesis (tumor formation)• Activate oncogenes• Inhibit tumor suppressor genes

Genome Editing

• Zinc finger Nucleases

• TALENs (transcription activator-like effectors (TALEs) nucleases)

• Zinc finger Nickases

Xanthomonas, producer of TALE proteins

NHEJ

HDR (homology-directed repair)