An improved self-deleting retroviral vector derived from avian leukemia and sarcoma virus

Abstract

We have previously developed a self-deleting avian leukosis and sarcoma virus (ALSV)- based retroviral vector carrying an additional attachment (att) sequence. Resulting proviruses underwent deletion of viral sequences and were flanked either by two LTRs (LTRs proviruses) or by the additional att sequence and the 3' LTR (att proviruses). Herein, we have tried to increase (1) the self-deleting properties of this vector, either by raising the selection pressure applied on target cells or by optimizing the size of the internal att sequence, (2) the titer of the vector by deleting or inverting some viral sequences. Moreover, a new type of provirus flanked by att sequences at each end was isolated. Finally, under specific conditions, 100% of proviruses had internal sequences deleted, and as many as 92-100% of proviruses were no longer mobilizable by a replication-competent virus. The inactivation procedure achieved here might improve the biosafety of retroviral vectors.

Gene Transfer into Mammalian Cells Using a Self-Deleting Avian Leukemia and Sarcoma Virus-Based Vector

“…1 b): (a) clones flanked by the additional att site in 5 and the LTR in 3 (proviruses H2-H3-H5-H6-H7-H11-H15 and H17) are grouped into class I. Proviruses H10 and H13 may also be classified into class I, but their inclusion in class II cannot be excluded, (b) clones displaying the full-length structure of the parent vector (class III): only one provirus (H16) exhibits the NP3Catt vector structure in full length, (c) clones displaying a rearranged structure (class IV): two proviruses (H8 and H14) present a permuted structure. A rearranged structure of this type has already been observed with the NP3Catt vector [10,11] and was assumed to result from autointegration events [20] . No class V clone (att-att) was found, but these clones are rare even in avian cells (only one such clone observed to date) [11] .…”

“…The presence of the 3 LTR is checked by PCR amplification using K2 and invR primers specific to the 3 noncoding region and to the LTR, respectively (data not shown). Secondly, the structure of the 5 region is further deter-A Novel Self-Deleting Retroviral Vector mined by PCR: (a) connection of the neo gene to the puro transcriptional unit in the neo-positive proviruses is checked by PCR using the N129 and O4 primers, which hybridize the neo and SV40 sequences, respectively, (b) the presence of the 5 LTR is monitored by using the R/ N859 pair of oligonucleotides (R is specific to LTR, N859 hybridizes neo), (c) clones displaying a rearranged structure are revealed with N130 and O4 primers (or invR/O6 [11] ). Thirdly, for neo-negative proviruses, an inverse PCR approach [10] is used to determine the host-viral DNA junctions ( fig.…”

“…The structure of the proviruses was examined by subjecting genomic DNA from each clone to molecular analysis, using a procedure previously developed on avian cells [10,11] and described in figure 1 b. Firstly, the overall structure of the provirus is checked by Southern blot and PCR. The 5 and 3 viral-host DNA junction fragments are revealed using the Hin dIII enzyme and hybridization with a neo or a puro probe.…”

“…1 a) is an avian erythroblastosis virus (AEV)-derived vector that carries the neo gene controlled by the viral LTR and the puro gene controlled by an internal SV40 promoter. An internal att sequence has been inserted between the two selectable genes [10,11] . The inefficient viral entry of the avian vector into human cells was circumvented by introducing the quail tv-a gene (encoding the receptor for ALSV of subgroup A) into Hela cells.…”

“…The transcriptional inactivation of the LTRs in the provirus enables an internal promoter to regulate expression of the transgene and reduces the risk of activating cellular genes. Secondly, self-deleting vectors have been developed to achieve the removal of the packaging sequence and/or drug resistance gene from the target cell: (a) the recombination system from bacteriophage P1 was used in Cre/ loxP vectors to excise the viral sequences located between two loxP target sites [8] ; (b) the high frequency of directrepeat deletion during reverse transcription was used in E-vectors to efficiently delete the packaging sequence and the selectable gene from SNV or MLV vectors [9] ; (c) we have previously described an avian leukosis and sarcoma viruse (ALSV)-based retroviral vector, containing an additional integration sequence (att) in an internal position and able to self-delete most of the 5 genome (including the 5 LTR, ⌿ sequence and selectable gene) [10,11] . Under specific conditions of selection, the internal sequences ( ⌿ sequence and/or selectable gene) were deleted in 100% of proviruses and as many as 92-100% of proviruses could no longer be mobilized by a replication-competent virus [10,11] .…”