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Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression

Abstract

Introduction of the post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) into the 3′ untranslated region of retroviral and lentiviral gene transfer vectors enhances both titer and transgene expression. Optimal use of the PRE is often necessary to obtain vectors with sufficient performance for therapeutic applications. The enhancing activity of the PRE depends on the precise configuration of its sequence and the context of the vector and cell into which it is introduced. However, data obtained in the context of WHV-associated hepatocellular carcinomas suggests that the PRE might potentially contribute to tumorigenesis, especially if encoding a truncated version of the WHV X protein. Oncogenic side effects of lentiviral vectors containing the PRE have reinforced these safety concerns, although a causal role of the PRE remained unproven. Here, we demonstrate that PRE mutants can be generated that are devoid of X protein open reading frames (ORFs) as well as other ORFs exceeding 25 amino acids, without significant loss of RNA enhancement activity. Furthermore, the X protein promoter could be deleted without compromising the enhancement of vector titers and transgene expression. Such a modified PRE sequence appears useful for future vector design.

Main

The post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) represents a powerful tool for improvement of 3′ RNA processing.1, 2 When introducing this orientation dependent RNA element into the 3′ untranslated region (3′ UTR) of gammaretroviral or lentiviral vectors, both vector titer and transgene expression can be enhanced.1, 3, 4 The mechanism of action involves coupling of the mRNA to CRM1-dependent nuclear export.5 This pathway is typically reserved for proteins but can also be exploited by other viral export machineries, as exemplified with Rev-dependent export of lentiviral sequences containing the Rev-responsive element (RRE).6, 7, 8 As the PRE needs to be positioned in the 3′UTR for optimal performance and also enhances titers of lentiviral vectors in the presence of Rev/RRE, additional effects on 3′ RNA processing such as efficient termination or polyadenylation are likely.2 As polyadenylation promotes translation,9 the PRE might even improve transgene expression.3, 4 While both Hepadnaviridae (to which WHV belongs) and Retroviridae depend on reverse transcription of RNA into DNA during viral replication, the PRE is not known to be involved in this process. Owing to its modular configuration, the PRE is also of interest for transgene expression in DNA-based vectors such as those derived from adeno-associated viruses.10 Very recently, the PRE was shown to overcome the negative effects of CTG repeats by counteracting nuclear retention of mRNAs.11

The extent to which the PRE enhances transgene expression depends on its sequence, the fragments used and the specific setting determined by vector context and the target cell.1, 2, 3, 4, 12, 13 Optimal use of the PRE can increase both titer and expression of gammaretroviral or lentiviral vectors by a factor of 2–10, which is often necessary to obtain vectors with sufficient performance for therapeutic applications.14, 15, 16 However, data obtained in the context of WHV-associated hepatocellular carcinomas suggests that sequences overlapping with the PRE might potentially contribute to tumorigenesis, especially if encoding a C-terminally truncated version of the WHV X protein.17 The C-terminally truncated X protein has a size of 70 amino acids, thus lacking 80 amino acids of the wild-type C-terminus. The precise function of the C-terminally truncated X protein is controversial, with some evidence reported for an antiapoptotic role.17 In addition, the induction of liver cancer by WHV appears to require insertional mutagenesis via insertion of WHV next to the N-myc gene.18 Importantly, the C-terminally truncated X protein lacks the majority of the transactivation domain, which recently has been shown to be necessary to enhance replication of hepatitis B virus.19 The open reading frame (ORF) for the ‘enigmatic’ X protein starts in the 3′ half of the PRE, and the corresponding transcript is initiated from a cryptic promoter contained in its 5′ region.1, 17, 20 In hepatic cells, the activity of this cryptic promoter heavily depends on two viral enhancers, one of which is located 3′ of the sequence typically used for vector construction.1, 21

Recently, induction of murine hepatocellular carcinomas has been observed following fetal delivery of lentiviral vectors based on an equine lentivirus containing a PRE that encodes a truncated X protein; however, in a control group injected with HIV-based lentiviral vectors no tumor development was observed so far, irrespective of the type of PRE used.22 Although a causal role of the PRE remained speculative, these data have triggered further safety concerns in the context of human gene therapy.23

To minimize potentially harmful effects of the PRE, we constructed a PRE version that is devoid of the X protein promoter and any residual ORFs, but still retains the beneficial effect on titer and transgene expression.

Based on the work of Donello et al.1 and computer predictions of RNA secondary structures, we have previously designed three PRE fragments (originally termed α, β and γ) and evaluated their function in retroviral vectors.4 To avoid confusion with the domain nomenclature proposed by Donello et al.,1 we here refer to the short fragment as aPRE (450 bp), the medium-sized as bPRE (600 bp) and the long as cPRE (900 bp). The cPRE fragment contains the majority of the X protein ORF. All PRE variants except the aPRE contain the X protein promoter (Figure 1). In comparison to these fragments, the PRE used in most state-of-the-art lentiviral vectors3 lacks the first 60 nucleotides but contains parts of the N-terminal ORF of the X protein (LPRE, Figure 1a). Although both the aPRE and the bPRE sequence are devoid of X protein sequences, they still contain several ORFs that encode peptides of more than 25 amino acids of unknown biological function, the largest of them in frame within the WHV polymerase ORF. These peptides, even if expressed at low levels, could be immunogenic.24

Figure 1
figure1

Schematic design of WPRE elements and vectors. (a) The post-transcriptional regulatory element (PRE) from Woodchuck Hepatitis Virus B (WHV) is drawn as a gray bar; nucleotide numbering is according to Genbank accession no. J02442. Five different PRE fragments have been used in the current study. Based on secondary structure predictions, three different PRE fragments were tested: aPRE (450 nt.), bPRE (600 nt.) and cPRE (900 nt.).4 The PRE fragment used in lentiviral vectors (nt. 1093–1684) was termed LPRE. The partially overlapping open reading frame (ORF) for the WHV X protein is drawn as a thick black line. The X protein promoter is depicted as a box (P). All four ATGs (marked by asterisks in the original sequence), which are followed by an ORF encoding more than 25 amino acids were mutated and this construct was named bPRE4*. The oPRE is derived from bPRE4* and is completely devoid of the X protein promoter. (b) Gammaretroviral and lentiviral vectors used in the present study are shown in their configuration after integration. Gammaretroviral vectors are based on murine leukemia virus,4 lentiviral vectors on human immunodeficiency virus.3, 25 Gammaretroviral LTR-driven vectors (MP110, MP71), gammaretroviral SIN vectors (Sin11.CMV) and lentiviral SIN vectors (LentiCMV) are depicted. MP stands for myeloproliferative sarcoma virus.28 The leader region of MP110 is devoid of any splice signals in order to evaluate effects of the PRE independently of splicing. In contrast, MP71 contains a splice-competent intron and a destabilized GFP version (d2GFP). The gammaretroviral and lentiviral self-inactivating vector (Sin11.CMV and LentiCMV, respectively) lack the entire enhancer–promoter of the U3 region15 and use the internal CMV promoter for expression in the target cells. In addition, the lentiviral SIN vector contains a Rev-responsive element (RRE) and a central polypurine tract (cPPT; not shown) in the leader region. The different PRE variants are located in the 3′ UTR. Primer binding site (θ), splice signals (SD, SA) and packaging signal (Ψ) are marked. The probes used for the Northern blots are shown as black lines. Details on the cloning procedures are available on request (schambach.axel@mh-hannover.de).

When introduced into LTR-driven gammaretroviral vectors (Figure 1b), the length of the PRE corresponded to its augmenting activity on retroviral RNA expression (Figure 2a). Accordingly, the largest element (cPRE) led to the strongest increase in protein expression (Figure 2b and Table 1). Nevertheless, the bPRE accounted for 74% of the effects of the cPRE, indicating that the 3′ part of the PRE including the X protein ORF is not absolutely required to enhance transgene expression (Table 1). In addition, we tested a version of the cPRE carrying a mutation in the X protein ORF ATG in comparison with the regular cPRE and found a slight increase in titer but unaltered gene expression in retroviraly transduced murine fibroblasts (data not shown). In line with this conclusion, Northern blot data did not reveal evidence for a cryptic promoter activity in Jurkat T cells transduced with cPRE-containing retroviral vectors under conditions of high multiplicity of infection (data not shown).

Figure 2
figure2

Increase in gene expression mediated by the PRE depends on length of the fragment used. (a) SW480 cells were transduced with retroviral vectors (MP110-based) containing different PRE fragments. A 48 h post-transduction total RNA was prepared as previously described4 and analyzed by Northern blot using a 3′ UTR probe indicated in Figure 1b. The blot was reprobed with a GAPDH-specific probe as a loading control. Molecular weight standard is given on the left. Note that transduction efficiencies of cells transduced with the indicated vectors varied as stated in Table 1. (b) To determine the enhancement on gene expression at the protein level, SW480 cells were transduced with retroviral vectors harboring different PRE fragments as indicated and described in Figure 2a and analyzed by FACS. The mean fluorescence intensity (MFI) is given on the left.

Table 1 Enhancement of transgene expression with different PRE fragments

However, other ORFs present in the PRE could be translated by reinitiation of translation following ribosomal detachment at the translational stop codon of the preceding transgene cassette. Therefore, we explored whether ORFs located in the bPRE sequences could be deleted without reducing its beneficial effect on transgene expression and viral titer. To this end, we have created the bPRE4* version, by mutating the start codons of all ORFs larger than 25 amino acids including one in frame with the WHV polymerase ORF25 (Figure 1a). Since the bPRE4* still contains the X protein promoter, we also mutated the mapped 21-nucleotide promoter domain (nt. 1482–1502, corresponding to Genbank acc. no. J02442).20 We generated three variants with either nt. 1482–1491 deleted, the most critical nt. 1490/91 mutated (TG → CA) or a complete promoter deletion. Among these promoter variants the complete promoter deletion was the most effective (data not shown) and theoretically safest variant. This optimized PRE (termed oPRE; Figure 1) and bPRE4* were introduced into gammaretroviral SIN vectors (Figure 1),25 lentiviral SIN vectors and conventional LTR-driven gammaretroviral vectors. Following production by transient transfection, murine fibroblasts (SC-1) were transduced with the different constructs.

In the retroviral SIN vector context, the mutant PRE fragments led to an increase in titer (five-fold) and in protein expression (two-fold) compared to the control (Figure 3a). Moreover, we found that the LPRE fragment typically used in lentiviral vectors did not perform as well in the gammaretroviral SIN context. Northern blot analysis confirmed these data (Figure 3b). Interestingly, the RNA detected using different PRE fragments showed a variable length. A possible explanation could include different polyadenylation. We also tested the different PREs in hepatic cell lines that are more related to the natural target cells of hepadnaviridae, using Hepa1.6 cells (murine hepatoblastoma line, Figure 3c and d) and Huh7 cells (human hepatoblastoma; data not shown). As example for hematopoietic cells we also included Jurkat T cells in our analyses (data not shown). In all cases, bPRE4* and oPRE were equally efficient in enhancing both titer and gene expression of gammaretroviral SIN vectors.

Figure 3
figure3

ORF-mutated and promoter-deleted versions of the PRE are still functional. (a) The PRE fragments (oPRE, bPRE4*, LPRE) were inserted into the 3′UTR of self-inactivating gammaretroviral (SIN) vectors bearing identical transgene cassettes driven by an internal CMV promoter.26 SC-1 murine fibroblasts were transduced with the indicated vectors at a low multiplicity of infection (transduction efficiency <35%) to ensure the presence of a single vector copy in the majority of transduced cells. At 5 days after transduction analysis was performed by FACS and the MFI of the GFP-positive cells was determined. Vector titer is indicated on the right side. Error bars were calculated from three independent experiments. (b) Northern blot analysis of transduced SC-1 cells (<35% pos. cells) according to (a). Analysis was performed as in Figure 2a. A probe corresponding to the GFP cDNA was used (probe II in Figure 1b). RNA species are marked on the right. (c) Titer and expression analysis of gammaretroviral SIN vectors habouring different PRE fragments in murine hepatoblastoma cells (Hepa1.6). Mean values and s.d. of three independent experiments are shown. (d) Northern blot analysis of Hepa1.6 cells transduced with the indicated constructs. Preparation was performed as in Figure 2a. RNA species are named on the right. (e) Lentiviral vectors bearing the same PRE versions as in (a). Analysis according to (a) and mean values and s.d. of three independent experiments are shown. (f) Titer and expression analysis of LTR vectors containing different PRE fragments. Titer determination was performed on SC-1 cells and d2GFP expression was analyzed by FACS. Mean values and s.d. of three independent experiments are shown.

In lentiviral SIN vectors, oPRE and LPRE were equally efficient (Figure 3e). The influence of the modified PREs on gene expression and vector titer was also tested with the LTR-driven gammaretroviral vector MP71 (Figure 1b). To allow a better correlation between fluorescence intensity and level of transgene expression, we used the destabilized GFP (d2GFP). When inserted into the 3′ UTR of MP71, both bPRE4* and oPRE increased titer (6–8-fold) and enhanced transgene expression up to two-fold (Figure 3f). In contrast, the LPRE had no beneficial effect on protein expression and only a very minor effect on titer (Figure 3f). Taken together, these experiments show that the oPRE preserved PRE function, in the context of different gammaretroviral and lentiviral vectors.

Our data confirm that the PRE can be divided into functional modules as shown by Donello et al.1 We created a functional PRE fragment devoid of any remnants of the X protein and all ORFs larger than 25 amino acids by mutating residual ATGs. To avoid any unwanted protein expression this bPRE4* construct was further modified by deleting the X protein promoter. This element (oPRE) is sufficient to enhance titer and gene expression in gammaretroviral SIN15, 26 and LTR-driven vectors as well as in the context of lentiviral SIN vectors.27 The oPRE or similarly designed fragments may represent useful tools for future vector development and clinical applications.

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Acknowledgements

We thank R Seyd and M Id for excellent technical assistance. We would like to thank H Schaller, U Protzer and H-G Kräusslich for helpful suggestions. This work was supported by Grants of the European Union (CONSERT, LSHB-CT-2004-005242) and the Deutsche Forschungsgemeinschaft (DFG BA 1837/4).

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Correspondence to T Giroglou.

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Schambach, A., Bohne, J., Baum, C. et al. Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression. Gene Ther 13, 641–645 (2006). https://doi.org/10.1038/sj.gt.3302698

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Keywords

  • post-transcriptional regulatory element
  • X protein
  • gamma retroviral vector

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