Retroviral vectors produced in the cytoplasmic vaccinia virus system transduce intron-containing genes

Abstract

Introns and polyadenylation (pA) sites are known to improve transcript stability and nuclear-cytoplasmic transport and are normally present in efficient gene expression vectors. Standard retroviral vectors, however, do not allow the inclusion of such sequence elements, as mRNA processing at internal splice and pA sites interferes with the production of functional full-length vector genomes. In this report we examined the capability of hybrid vaccinia/retroviral vectors to transduce complex gene cassettes with nuclear RNA processing signals within the retroviral genome. A retroviral vector was constructed that contains a gene of interest (the human coagulation factor IX [FIX] cDNA), including an intron and an internal pA site. The modified proviral vector genome was cloned downstream of a vaccinia virus promoter and was inserted into the vaccinia virus genome. Infection of a packaging cell line with the recombinant vaccinia virus vector resulted in secretion of retroviral particles at average titers of 10(5) CFU per ml of cell culture supernatant. Due to the cytoplasmic transcription and the nonrecognition of nuclear transcription signals in the vaccinia virus system, full-length transcripts were obtained that still contained the intron. In the retrovirally transduced cell lines the FIX transcripts were terminated at the internal pA site. The transcripts were quantitatively spliced, and FIX was secreted. Recombinant cell lines with stable single-copy inserts containing sequence elements necessary for efficient gene function could be generated. Thus, a relatively simple cytoplasmic system for the generation of complex retroviral vectors is described. Retroviral vectors transducing intron-containing gene cassettes may play a further role in gene therapy applications.

FIG. 1.

Schematic representation of the genetic elements in the viral genomes and transduced cells. (A) The modified retroviral transcription unit in the vaccinia virus (VV) vector. A vaccinia virus early promoter (bold arrow) is fused to the repeat region (R) of the LTR followed by the U5 region, the packaging signal, the small SV40 intron (i), and the FIX open reading frame, which is terminated by the internal pA site (pA1). The second transcription unit, consisting of the SV40 promoter and the neomycin open reading frame, is followed by the 3′ LTR that includes the terminal pA site (pA2). The SacI restriction site (S) present in the U3 region was used for genomic characterization. A vaccinia virus early transcription stop signal (*) is located immediately downstream of the second pA site. (B) The modified retroviral vector produced in the cytoplasmic vaccinia virus system retains the intron. The RNA genome has a CAP structure at the 5′end and a poly(A) tail (AAAAA) at the 3′ end. (C) In the transduced host cell, the LTR promoter (located in the U3 sequence) is reconstituted during RT and the integration process driving the FIX gene cassette.

FIG. 2.

Western blot analysis of the FIX expression in the transduced 3T3 cell clones (A) and in the transduced CHO cell clones (B). (A and B) Lane 1, marker proteins; lane 2, FIX standard purified from plasma; lane 3, cell culture supernatants from nontransduced parental cell lines; lanes 4 to 6, cell culture supernatants from transduced cell clones. The arrowheads on the right point to the FIX band.

FIG. 3.

Genomic analysis of the DNA of the cell clones by PCR for the presence of the FIX sequences (A) and for the neomycin selection marker (B) and by Southern blot for presence of the FIX and intron sequences (C). Lane 1, size markers (M); lane 2, viral DNA of the hybrid vaccinia virus vR-Xi9pASN; lane 3, negative control DNA of NIH 3T3 cells; lanes 4 to 6, DNA of the transduced clones 3T3FIX#1.1, 3T3FIX#2.4, and 3T3FIX#2.5. The numbers on the right are the sizes of the bands in base pairs.

FIG. 4.

Analysis of the transcripts of the transduced cell clones and the vaccinia virus-infected cells by RT-PCR (A) and outline of the primers (P1-P2) and the template used (B). (A) Lane 1, size markers (M); lane 2, RNA of cells infected with the hybrid virus vR-Xi9pASN; lane 3, negative control RNA of NIH 3T3 cells; lanes 4 to 6, RNA of the transduced clones 3T3FIX#1.1, 3T3FIX#2.4, and 3T3FIX#2.5. The numbers on the right are the sizes of the bands in base pairs.

FIG. 5.

RT-PCR analysis of the transcripts of a transduced cell clone spiked with unspliced vaccinia virus RNA. Lane 1, marker (M); lane 2, RNA of the NIH 3T3 negative control; lane 3, RNA of cells infected with the recombinant vaccinia virus (rVV) vR-Xi9pASN; lanes 4 to 7, RNA of the transduced cell clone 2.4 spiked with the indicated amounts of unspliced viral RNA. The numbers on the right are the sizes of the bands in base pairs.

FIG. 6.

Schematic representation of the retroviral transcription unit and the expected transcripts (t1 to t3) in the transduced host cells. SD1, splice donor; SA, splice acceptor. The two pA sites (pA1 and pA2) are indicated by arrows. The sizes of the predicted transcripts are on the right.

FIG. 7.

Northern blot analysis of the transcripts derived from the transduced cell clones and the controls. (A) A schematic representation of the binding regions of probes 1 and 2. Probe 1 is complementary to the region upstream of the AATAAA signal (underlined) of the FIX transcripts. Probe 2 is complementary to the region downstream of the processing signal. (B and C) The Northern blots obtained with probe 1 (B) and probe 2 (C). The transcripts (t1-t2, left side) and their respective sizes in kilobases (right side) are shown. Lanes 1 to 3, analysis of RNAs from transduced cell clones 3T3FIX#1.1, 3T3FIX#2.4, and 3T3FIX#2.5; lane 4, negative control RNA from NIH 3T3 cells; lane 5, positive control RNA from a classically transfected and amplified CHO cell clone expressing human FIX; lane 6, control RNA from CV-1 cells infected with the vaccinia virus vR-Xi9pASN. (D) A control blot obtained with a GAPDH probe confirming equal RNA loading of the gel.

FIG. 8.

RT-PCR of transcripts terminating at the internal pA site and sequence analysis of cDNAs. (A) Schematic outline of the primers and the template used to map pA. (B) Agarose gel electrophoresis showing the results of the PCR analysis. Lane 1, size markers; lane 2, RNA of cells infected with the hybrid virus vR-Xi9pASN; lane 3, negative control RNA of NIH 3T3 cells; lanes 4 to 6, RNA of the transduced clones 3T3FIX#1.1, 3T3FIX#2.4, and 3T3FIX#2.5. The number on the right is the size of the bands in base pairs. (C) Sequence of plasmid control pR-Xi9pASN and the cDNAs derived from the internally terminated transcripts.