Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI.
BACKGROUND
A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O6-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting.


RESULTS
All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was approximately 4 times greater than that of an IRES based vector.


CONCLUSIONS
The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.
Chinnasamy
Dhanalakshmi
D
Vince Lombardi Gene Therapy Laboratory, Immunotherapy Program, Aurora St. Luke's Medical Center, Milwaukee, WI 53215, USA. dhana.chinnasamy@aurora.org
Milsom
Michael D
MD
Shaffer
James
J
Neuenfeldt
James
J
Shaaban
Aimen F
AF
Margison
Geoffrey P
GP
Fairbairn
Leslie J
LJ
Chinnasamy
Nachimuthu
N
D004686:Encephalomyocarditis virus / Q000235:genetics; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes
C090460:HOXB4 protein, human; D018398:Homeodomain Proteins; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase