From retroviral vector production to gene transfer: spontaneous inactivation is caused by loss of reverse transcription capacity.
BACKGROUND
The loss of gene transfer capacity in retroviral vectors constitutes a major disadvantage in the development of retroviral vectors for gene therapy applications. In the present work the loss of a vector's capacity to perform reverse transcription was studied as a possible explanation for the low stability of retroviral vectors from the production stage to the target cell gene transfer event.


METHODS
Inactivation studies were performed with murine leukemia virus vectors at 37 degrees C and several residual activities were tested, including viral infectivity, reverse transcription capacity, reverse transcriptase (RT) activities and viral RNA stability.


RESULTS
The results indicate a high correlation between loss of infectivity and the capacity of the virus to perform the initial steps of reverse transcription. To further understand the thermosensitivity of the reverse transcription process, the two enzyme activities of RT were investigated. The results indicate that, although the inactivation rate of the DNA polymerase is faster than that of RNase H, the decline of these two enzyme activities is significantly slower than that of reverse transcription. Also, viral RNA stability is not implicated in the loss of the virus capacity to perform reverse transcription as the rate of viral RNA degradation was very slow. Furthermore, it was observed that the amount of viral RNA that entered the cells decreased slowly due to viral inactivation at 37 degrees C.


CONCLUSIONS
The reverse transcription process is thermolabile and this sensitivity determines the rate of retroviral inactivation. Strategies targeting stabilization of the reverse transcription complex should be pursued to improve the applicability of retroviral vectors in gene therapy studies.
Carmo
M
M
IBET/ITQB, Oeiras, Portugal.
Panet
A
A
Carrondo
M J T
MJ
Alves
P M
PM
Cruz
P E
PE
D002460:Cell Line; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D016679:Genome, Viral; D006801:Humans; D009052:Leukemia Virus, Murine / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D020871:RNA Stability; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D048348:Reverse Transcription; D016914:Ribonuclease H / Q000235:genetics; D038301:Virus Inactivation
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H