mental signal normalized to the H 2 B signal for the conespond- ...... H. Velander, Timothy K. Lee, Dorothea H. Scandella, Francis C. Gwazdauskas, James.
H U M A N G E N E T H E R A P Y 6:469-479 (April 1995) Mary A n n Liebert, Inc., Publishers
S e q u e n c e s W i t h i n t h e C o d i n g R e g i o n s o f Clotting F a c t o r V I I I and C F T R
B l o c k Transcriptional E l o n g a t i o n
DWIGHT D. KOEBERL, CHRISTINE L. HALBERT, ANTON KRUMM, and A. DUSTY MILLER
ABSTRACT The clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs have dramatically reduced levels of expression compared to clotting factor I X (FIX) and other c D N A s (100 and 1,000-fold lower, respectively), w h e n produced in cells by using an expression vector. Part of the inhibitory signal in the FVIII c D N A has been localized to a 1.2-kb inhibitory sequence (FVIII INS), which decreased steady-state R N A levels from a retroviral vector by 30- to lOO-fold. A n analysis of R N A degradation indicated that the FVIII I N S vector R N A is relatively stable. Nuclear run-on experiments with the FVIII I N S vector demonstrated a low signal for FVIII, in contrast to the high signal for a F I X vector. T h e low signal for FVIII I N S w a s not due to a decrease in transcriptional initiation. Thus, FVIII expression is reduced through a block to transcriptional elongation, as has been found in c-myc and other genes. W e show that the inhibitory effect of F V n i I N S is orientation dependent with regard to the promoter. In addition, the inhibitory effect is position dependent, because expression of FVIII I N S sequence increased w h e n it was m o v e d 1 k b further from the promoter in a retroviral vector. Similar results were observed by using a retroviral vector for expression of the C F T R c D N A . T h e C F T R retroviral vector produced 1,000-fold decreased steady-state R N A levels, compared to the parent vector. Nuclear run-on analysis with the C F T R vector revealed a block to transcriptional elongation within the C F T R c D N A . T h e presence of blocks to transcriptional elongation within the FVIII and C F T R c D N A s complicates efforts to produce high levels of these proteins for therapeutic purposes and to develop high-titer retroviral expression vectors for h u m a n gene therapy.
OVERVIEW S U M M A R Y
the expression ofthe C F T R c D N A is regulated at the level of transcriptional elongation. Decreased expression of the Koeberi et al. analyzed the control of expression for FVIII the and C F T R c D N A s from retroviral vectors compliF V H I and C F T R c D N A s in the context of retroviral expres- cates efforts toward human gene therapy. sion vectors by utilizing R N A stability and nuclear run-on assays. Vector R N A containing FVIII INS was stable for at least 6 hr following inhibition of R N A synthesis by using INTRODUCTION actinomycin D. Nuclear run-on analyses revealed that F V n i INS vectors generate a higher prevalence of upstream transcripts compared to the FVIII INS sequence, indicating Factor VIII (FVIII) is primarily synthesized in the liver, but FVlll m R N A levels in hepatocytes are extremely low, the presence of a block to transcriptional elongation. The block to transcriptional elongation in FVIII INS was found comprising only 0.001% of m R N A in the liver, or 7,000-fold to be orientation-dependent. The FVIII INS inhibitory se- less than the amount of albumin m R N A (Wion et al., 1985). quence was localized to several elements that have an addi- The FVlll c D N A is expressed at much lower levels from a tive inhibitory effect on expression. Like the FVIII c D N A , plasmid expression vector than the von Willebrand's factor or
Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, W A 98104.
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dihydrofolate reductase (DHFR) cDNAs (Kaufman et al., Here w e have examined the contribution of these factors to 1989). The FVIII c D N A is also expressed at dramatically low decreased FVIII and C F T R R N A production. levels from retroviral expression vectors (Hoeben et al., 1990; Israel and Kaufman, 1990; Lynch et al, 1993). Markedly decreased expression of the FVlll coding regions, compared to MATERIALS AND METHODS other coding regions, has been observed for all systems in Cell culture which it has been analyzed. Clotting factor IX (FIX), adenosine deaminase ( A D A ) , and PA317 amphotropic retrovirus packaging cells (Miller and other c D N A s are expressed at high levels from retroviral vecRosman, 1989) and N1H-3T3 thymidine kinase-negative tors (Miller, 1992; Palmer et al., 1989, 1991), whereas the ( T K - ) cells (Wei etal., 1981) were cultured in Dulbecco-Vogt F V U I c D N A is expressed at very low levels from these vectors modified Eagle's medium ( D M E M ) with high glucose (4.5 (Hoeben etal., 1990; Israel and Kaufman, 1990; Lynch etal., grams/liter) supplemented with 1 0 % fetal bovine serum and the 1993). For example, human fibroblasts transduced with a retroantibiotics penicillin, streptomycin, and amphotericin B. viral vector containing the FIX c D N A produced approximately 3 p,g of FIX protein per million fibroblasts each day (Palmer et al., 1989), and human fibroblasts transduced with a FVIII ret- Construction of FVIII and CFTR retroviral vectors roviral vector produced only about 40 ng of FVlll protein per million cells each day (Lynch et al., 1993). In an attempt to The FVIII vectors pLBHSN, pLPSSN, pLBBSN, and develop an animal model for gene therapy, fibroblasts were p L S H S N (Lynch etal., 1993), the FIX vector p L I X S N (Palmer transduced with a FVin retroviral vector in vitro and trans- et al., 1989), and the L X S N vector (Miller and Rosman, 1989) planted into irrunune-deficient mice, but no human FVIII could have been described. The retroviral vector nomenclature uses be detected in vivo (Hoeben et al., 1993). The FVIII c D N A in the prefix "p" to denote the plasmid form; the prefix is dropped retroviral vectors has the activation peptide, or B domain, de- to indicate the viral form. The vector p L B H a S N was made by leted to reduce its size due to space constraints, but the synthe- creating blunt ends for the Eco Rl-Hpa 1 FVIII INS fragment of sis of active FVlll protein from plasmid or retroviral vectors is p L B H S N , and inserting the fragment in reverse orientation in not impeded by this deletion (Burke et al., 1986; Eaton et al., the H p a 1 site of p L X S N . p L N B H was constructed by inserting 1986; Israel and Kaufman, 1990). FVIII R N A steady-state lev- die blunt Eco Kl-Hpa I FVlll INS fragment of p L B H S N in die els are reduced 100-fold from a FVlll retroviral vector com- forward orientation in the Nru I site downstream of the neo pared to the same vector expressing other c D N A s , and FVlll c D N A in the retroviral vector p L N L 6 (Miller and Rosman, vector titers are conespondingly reduced 100-fold compared to 1989). The vector p L D 2 S N was made by digestion of p L B H S N other vectors (Lynch et al., 1993). A large part of the inhibitory with Bst XI and then incubating with Bai 31 exonuclease, effect of the FVlll c D N A on expression from a retroviral vector followed by Eco Rl digestion to define the 5' end, incubation has been localized to a 1.2-kb fragment, derived from the A 2 with Klenow D N A polymerase to create blunt ends, and ligaand A 3 domains, which decreased steady-state R N A levels tion of the resultant vector to produce a vector with a 5' deletion 100- to 200-fold and decreased vector titers 10-fold (Lynch et in the FVlll INS insert. The C F T R vector p L C F S N was conal., 1993). The mechanism by which the FVIII c D N A de- structed as described (Olsen et al., 1992). Vectors containing portions ofthe FVIII INS sequence (numcreases R N A and protein expression from expression vectors bering system of Toole et al., 1984) were constructed by insertremains to be established. The cystic fibrosis transmembrane conductance regulator ing the following fragments into the retroviral vector p L X S N (CFTR) c D N A also has low expression in vivo and in vitro. X H , Bst Xl-Hinc 11 (2,118-2,277, followed by 5,002-5,582) C F T R m R N A comprises less than 0.01% of m R N A in the lung X S , Bst Xl-Stu I (2,118-2,277, followed by 5,002-5,392) and pancreas, where its levels are highest (Riordan et al., X P , Bst Xl-Pst I (2,118-2,277, followed by 5,002-5,159). 1989). A C F T R retroviral expression vector had titers SO- to The FVIII sequences between positions 2,277 and 5,002 corre100-fold reduced compared to the parent vector, and steady- spond to the activation peptide and have been deleted (Lynch et state R N A levels in transduced cells were equivalent to approx- al., 1993). imately 0.01% of the poly(A) R N A (Riordan et al., 1989; D r u m m et al., 1990). The mechanism of inhibition by the Virus production C F T R c D N A has not been analyzed. Possible mechanisms for the control of steady-state R N A Transient virus production was assayed as described (Miller levels include the rates of transcriptional initiation and elonga- and Rosman, 1989; Lynch et al., 1993). Virus stock was pretion, R N A egress from the nucleus, and R N A degradation. The pared from a vector-producing P A 3 1 7 cell line by incubating efficiency of transcriptional elongation has recently emerged as medium for 16 hr with confluent layers of the cells. ThePA317/ an important mechanism for the control of expression, which L I X S N c7 and P A 3 1 7 / L B H S N c5 vector-producing packaging has been found within the adensonine deaminase ( A D A ) , c-fos, cell lines have been described (Palmer et al., 1989; Lynch et c-myc, and other genes (Spencer and Groudine, 1990). The al., 1993). N1H-3T3 cells were transduced with retroviral veceffect of a block to transcriptional elongation on steady-state tors, and individual colonies were selected. A Southem blot R N A levels can be significant; for example, the block to tran- revealed that the copy number for each vector in transduced scriptional elongation in the c-myc gene decreases the signal in N1H-3T3 cells was equivalent. Transduced NIH-3T3 and a nuclear run-on analysis for downstream sequences in differen- PA317 cells are pooled populations in which the retroviral tiated H L 6 0 cells 15-fold (Bentley and Groudine et al., 1986). vector is chromosomally integrated in a random manner.
FACTOR VIII AND CFTR TRANSCRIPTIONAL ELONGATION
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Nuclear run-ons
LBHSN LIXSN 0 0.5 1 3 0 0.5 1 3 6 hr Nuclear run-ons were performed as described previously; all experiments used a 150 inM KCl buffer (Groudine era/., 1981). After the DNase 1 treatment and phenol-chloroform extraction Vector of run-on transcripts, the nucleic acids were precipitated with ethanol and unincorporated nucleotides were removed by use of a Sephadex G 5 0 spin column. Labeled transcripts were hybridized to slot-blotted GeneScreen filters containing doublestranded D N A probes noted below. Double-stranded D N A probes were generated by restriction digest or P C R amplification with appropriate primers and purified from an agarose gel. After hybridization, the filters were treated for 30 min at 37°C with RNase A (10 p,g/ml), and washed twice in 1 x SSC, c-myc 1 % S D S at 55°C for 15 min each. Signals from blots were quantitiated with a Molecular Dynamics phosphorimager (Johnston et al., 1990). Probes included the S m a \-Eag 1 fragment of p L X S N (Psi-LTR), the Eag \-Eco RI fragment of beta-actin p L X S N (Psi), die B a m H\-Nco I fragment of p L X S N (SV40), the neo c D N A amplified from p L X S N (neo), the Eco Rl-Hpa I fragment of p L B H S N (INS), the Eco Rl-Bam HI fragment of L B H S N ( B M ) , the B a m HI-Hpa 1 fragment of L B H S N ( M H ) , FIG. 1. R N A stability analysis by transcriptional anest with die Bst Xl-Hpa I fragment of L B H S N (XH), the S' 2,108-bp actinomycin D. N1H-3T3 cells were transduced with viral vecEco RI fragment of the C F T R c D N A (CFl), the adjacent 3' tors from cloned PA317 packaging lines ( L B H S N c5 and l,45S-bp Eco Rl fragment of the C F T R c D N A (CF2), and the L I X S N c7), and exposed to actinomycin D (10 p.g/ml) for the FIX c D N A amplifled from pLIXSN with primers thatflankthe times indicated. Total R N A was harvested for a Northem blot. multiple cloning site (MCS). The number of Ts in the sense Probes included the neo c D N A , c-myc exon 1, and chicken strand of the relevant probes is as follows: Psi-LTR (88), Psi P-actin c D N A . The c-myc signal was not detectable after I hr, (171), S V 4 0 (48), neo (177), INS (343), FIX (477), CFl (488), which demonstrated the effectiveness of actinomycin D at causand C F 2 (398). Results are expressed as a ratio of the experi- ing transcriptional anest. mental signal normalized to the H 2 B signal for the conesponding blot, as measured by a phosphorimager, and conected for compared to an empty vector (Lynch et al., 1993). Here w e the relative T content of each probe. found that the steady-state levels of vector R N A from NIH-3T3 cells transduced with a FIX vector (LIXSN) were approximately 25-fold higher than for cells transduced with a FVIII INS vector ( L B H S N ) (Fig. 1). The rate of degradation of F V m Cytoplasmic a n d nuclear R N A preparation INS vector R N A relative to the FIX vector R N A was analyzed Nuclear RNA was prepared from frozen nuclei stored at to assess the role of R N A degradation in down-regulating FVin —70°C. Cytoplasmic R N A was prepared by centrifugation to INS expression. Vector R N A is transcribed from the 5' long remove nuclei in the presence of placental RNAase inhibitor terminal repeat (LTR) promoter and includes the c D N A insert, and 0.5% NP-40 (Sambrook etal., 1989). S V 4 0 promoter, neo gene, and 3' L T R in a single transcript (see vector diagram. Figure 3, below). A smaller neo transcript is generated by the intemal S V 4 0 promoter. Medium containing Transcriptional and translational arrests actinomycin D (10 p-g/ml) was added to NIH-3T3 cells transduced by the viral vectors L B H S N or LIXSN, which express Transcriptional arrest of subconfluent transduced N1H-3T3 FVIII INS or FIX, respectively. The full-length L B H S N R N A , cells was initiated by the addition of actinomycin D (10 p,g/ml) containing FVlll INS c D N A and neo, persisted for 6 hr without to the medium. Cells were scraped and total R N A was hardiminution, similar to the full-length L I X S N vector R N A , convested at time points from 30 min to 6 hr later. Cycloheximide taining the FIX c D N A and neo (Fig. 1). W h e n the same blot (10 p,g/ml) was added to the medium of subconfluent N1H-3T3 was hybridized to a P-actin probe, similar signals were seen in cells, and total R N A was harvested up to 5 hr later. all lanes, indicating approximately equal loading of R N A in all lanes (Fig. 1). In contrast, the signal for c-myc R N A disappeared after 1 hr, when the same blot was hybridized to a mouse c-myc exon 1 probe (Fig. 1). Disappearance ofthe c-myc signal RESULTS indicates that transcriptional anest occuned, and that labile R N A s would be expected to persist only during early time Steady-state RNA levels for a FVIII INS vector are not points. A smaller neo R N A , transcribed from the S V 4 0 proreduced due to R N A instability moter, persisted for the duration of the experiment, indicating A FVIII INS retroviral vector ( L B H S N ) consistently demon- the relative stability of this transcript. The vector R N A containstrated steady-state R N A at 100- to 2(X)-fold reduced levels ing FVIII inhibitory sequences is markedly more stable than the
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KOEBERL ET AL.
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F I G . 2. Analysis of total R N A , cytoplasmic R N A , and nuclear R N A from transduced N1H-3T3 cells. Total R N A , cytoplasmic R N A , and nuclear R N A were extracted from N1H-3T3 cells transduced by the indicated viral vectors. Northem blots were hybridized to neo and chicken p-actin probes as indicated. Signals were quantitated with a phosphorimager to compare steady-state R N A levels for various transcripts. The relative decrease of vector R N A for L B H S N vector R N A compared to L X S N vector R N A was consistent for all preparations. The neo signal for L B H S N is usually two- to three-fold reduced compared to other vectors. Transcripts intermediate in size between the full-length vector R N A and neo transcripts were observed for L B H a S N and, to a lesser extent, other vectors; w e believe that these bands represent vectors transcribed from reananged plasmids in pooled P A 3 1 7 packaging cells. Intermediate bands were almost nonexistence when the vector L B H S N was produced by a cloned P A 3 1 7 packaging cell line (Fig. 1). c-myc R N A , and has similar stability to the FIX vector R N A , indicating that R N A instability is not responsible for low FVlll INS steady-state R N A levels.
A block to transcriptional elongation within the FVIII INS decreases transcription compared to the FIX cDNA
RNA transport from the nucleus is not an obstacle to FVIII I N S expression
The relative levels of transcriptional initiation and elongation of F V I U INS compcued to other vector sequences and F I X was determined by nuclear run-on analysis, which quantitates the distribution of R N A polymerase II complexes along a particular sequence. Previously initiated nascent R N A molecules were elongated in the presence of radiolabeled U T P , and labeled R N A was isolated. Labeled transcripts from each preparation of nuclei were hybridized to unlabeled D N A probes slot-blotted to a nylon membrane, and the signal for each probe was quantitated with a phophorimager, normalized to a histone H 2 B or P-actin signal for that blot, and conected for relative U T P content. Nuclear run-on analyses were performed with NIH3T3 cells transduced with the L I X S N and L B H S N vectors. The signal for a FIX c D N A probe from the L I X S N nuclear ran-on was elevated 17- to 20-fold over the same probe in the L B H S N nuclear mn-on, demonstrating a high FlX-specific signal (Fig. 3A,B). The signal for the FVlll INS probe (INS) in die L B H S N nuclear run-on was equivalent to the signal for that probe in the L I X S N nuclear run-on, indicating that FVIII INS transcription was not above background and that a block to transcriptional elongation or low transcriptional initiation was responsible (Fig. 3A, B). The signal from the upstream probe Psi-LTR was high for all vectors assayed (Fig. 3). A similar signal for an upstream probe indicates that transcriptional initiation was not dramatically affected by FVIII INS, compared to FIX. The L I X S N signal was as high as that from die upstream Psi-LTR signal (when conected for U T P content of transcripts which hybridize to those probes), indicating that no significant block to elongation is present in die FIX c D N A (Fig. 3 A , B). Although L B H S N revealed a high level of transcriptional initiation, very little transcription of FVIII INS was detected. This is
The relative roles of nuclear processing and transport from the nucleus in the control of FVlll INS R N A expression were evaluated by comparing the steady-state levels of nuclear, cytoplasmic, and total R N A levels for L B H S N and other vectors. The steady-state levels of vector R N A for L B H S N were consistently decreased 30- to 50-fold compared to the parent vector, L X S N , in total, cytoplasmic, and nuclear R N A (Fig. 2). The paucity of L B H S N vector R N A in the nuclear fraction suggests that transport from the nucleus was not the cause for decreased expression of L B H S N . A vector expressing FVlll INS in an antisense orientation ( L B H a S N ) was constructed to assess the possible orientation dependence of the inhibitory effect of this sequence. Vector R N A levels were elevated 15-fold for the antisense FVlll INS, compared to L B H S N , in cytoplasmic and total R N A (Fig. 2). A vector expressing FVlll INS from a further downstream position was constructed ( L N B H ) , to assess the possible position dependence of the inhibitory effect of FVlll INS. The steady-state vector R N A level for L N B H was elevated six-fold relative to L B H S N in cytoplasmic and total R N A (Fig. 2). Splicing of FVIII INS vector R N A does not appear to play a role in the control of expression of FVlll INS, because no intemal splice sites are present in the FVlll c D N A . The unvarying ratio of vector R N A for L B H S N compared to L X S N in cytoplasmic, nuclear, and total R N A suggests that impaired nuclear egress does not prevent the appearance of FVIII INS vector R N A in the cytoplasm, and that nuclear processes other than splicing are responsible for down-regulating FVIII INS expression.
473
FACTOR VIII A N D CFTR TRANSCRIPTIONAL ELONGATION LBHSN
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- ^ c •mzzzzmmi t 1 kb Psi-LTR Psi SV40 FIG. 3. Nuclear mn-on analysis for L I X S N , L B H S N , and L B H a S N . A Nuclear mn-on analyses for N1H-3T3 cells transduced with the vectors L I X S N , L B H S N , and L B H a S N were performed. Each value represents averages for two independent nuclear mn-on analyses, except for INS in the L B H S N nuclear mn-on analysis, which represents four nuclear mn-on analyses. Signals were normalized to the H 2 B signal for each nuclear mn-on analysis and conected for the relative U T P content of each probe. The standard enor is indicated. Results were similar when signals were normalized to the p-actin signal for each blot. The background signal from NIH-3T3 cells was subtracted from vector probe signals (Psi-LTR, SV40, and neo). The background for each c D N A probe was the signal for that probe for a nuclear mn-on with another vector (i.e., the signal for the INS probe in the L B H S N nuclear mn-on was corrected for the background signal for INS in the L I X S N nuclear mn-on). B. A representative nuclear mn-on autoradiogram for each vector is shown. The S V 4 0 signal is not shown for all blots. C. The retroviral vector L X S N and the inserts for other vectors derived from L X S N are shown. Inserts conesponding to the H X c D N A (LIXSN) and FVlll INS ( L B H S N ) were cloned in the multiple cloning site ( M C S ) . Probes derived from the parent vector, L X S N , are indicated. The FVlll INS fragment consists of a 1.2-kb sequence within the FVlll c D N A , which is comprised of sequences from the A 2 and A 3 domains. Vector probes:
consistent with a block to transcriptional elongation wifliin FVIII INS. The possibility remains that transcriptional initiation for L B H S N is reduced two- to three-fold, compared to L I X S N . Smaller probes derived from FVIII INS ( B M , X H , and M H ; see Fig. 3C) did not detect a signal above background, indicating that the block to transcriptional elongation could not be localized to the 5' or 3' region of FVIII INS (not shown). The S V 4 0 promoter probe is located downstream of c D N A inserts in vectors derived from p L X S N , and consists of sequences upstream of the S V 4 0 transcriptional start site. The S V 4 0 probe demonstrated a high signal for the L I X S N vector relative to the background from untransduced NIH-3T3 cells, but the S V 4 0 signal from L B H S N (the F V I H INS-containing
vector) was relatively low (Fig. 3 A and data not shown). This difference presumably reflects higher readthrough transcription from the L T R promoter for LIXSN, and is consistent with a block to transcriptional elongation in FVIH INS. The relatively low signal for a probe downstream of FVIII INS confirmsfliepresence of a block to transcriptional elongation within FVIII INS. The block to transcriptional elongation within FVIII I N S is orientation d e p e n d e n t A transcriptional terminator sequence from the adenoviral major late promoter w a s found to decrease the signal for d o w n stream sequences in a nuclear m n - o n analysis in an orientation-
474
K O E B E R L E T AL.
T r a n s d u c e d packaging cells dependent manner (Connelly and Manley, 1989). To analyze possible orientation dependence for the FVlll INS inhibitory z 2 z z effect, F V I U INS was inserted into L X S N in an antisense orienCO CO z CO CO -^ tation (vector L B H a S N ) . Steady-state R N A levels for this vecLL LL (f) LL oLL CO o o o ^ 3 tor increased IS-fold over the vector L B H S N (Fig. 2). The _1 1 1 prevalence of steady-state R N A for the antisense FVlll INS • LCFSN vector vector (LBHaSN) was decreased only two- to three-fold compared to the L X S N vector (Fig. 2). T o conelate increased expression of the antisense FVlll INS vector with levels of transcription, a nuclear mn-on was performed with the L B H a S N vector. In contrast to the finding for the sense FVlll INS vector ( L B H S N ) , an intermediate signal for the INS probe was • LXSN vector detected above background (Fig. 3A, B ) . The INS signal for L B H a S N was approximately three-fold lower than the signal for the upstream Psi-LTR probe (Fig. 3A). The two- to threefold decrease in steady-state R N A levels for L B H a S N , compared to the parent vector L X S N , is consistent with the approximately three-fold lower signal in a nuclear mn-on analysis for the antisense FVlll INS compared to upstream vector sequences 2 hr exposure 20 hr exposure (Figs. 2 and 3A). The block to transcriptional elongation in FVlll INS is orientation dependent, as reflected by an increased FIG. 4. Total R N A from packaging cells transduced with signal in a nuclear mn-on for that sequence in the antisense L C F S N and L X S N vectors. Northem blots of total R N A from packaging cells and N1H-3T3 cells transduced with the vectors orientation. L C F S N and L X S N are shown. The probe for both blots was the neo gene. T w o exposures, for 2 hr and 20 hr, are shown for a Northem blot of total R N A from packaging cells transduced The C F T R c D N A contains a block to elongation that with L C F S N or L X S N from cloned PA317 packaging cells. markedly decreases downstream transcription Phosphorimager analysis ofthe L C F S N and L X S N vector R N A A retroviral vector containing the CFTR cDNA demonstrated signals from packaging cells revealed a 1,000-fold difference in low titers and steady-state R N A levels ( D m m m et al., 1990), steady-state levels for these R N A s . Ethidium bromide staining which suggests that an inhibitory sequence similar to FVIU INS ofribosomalR N A bands revealed that loading of each lane was might be present. The CFTR-containing vector, L C F S N , ex- approximately equivalent (not shown). pressed extremely low steady-state R N A levels in transduced cells. Vector R N A levels for packaging cells transduced with the L C F S N vector from cloned PA317 packaging cells were decreased 1,000-fold relative to packaging cells transduced with L X S N (Fig. 4). A 20-hr exposure of the Northem blot was multiple inhibitory sequence elements within F V U I INS, vecrequired to detect a weak L C F S N vector signal, compared to a tors that contained progressive deletions of the 5' and 3' termini strong signal for L X S N after 2 hr. W h e n analyzed in the same ofthe FVlll INS fragment were constmcted. After the deletion experiment, the Psi-LTR signal for cells transduced with of S' and 3' sequences from the F V U I INS vector ( L B H S N ) L C F S N was equivalent to that observed for other vectors, ininsert, a gradual increase in the expression of these vectors was cluding L B H S N and L I X S N (not shown). The signal for CFl, a observed; viral titer and steady-state R N A levels increased as downstream probe for thefirst2,108 bp of the C F T R c D N A , the size of the residual FVlll insert was decreased (Fig. 6 and gave at least a two-fold lower signal than the Psi-LTR probe Table 1). N o element was isolated that could account for the (Fig. SA, B). Moreover, a probe further downstream within the C F T R c D N A , C F 2 , had a signal that was not above back- majority of the inhibitory effect. Deletion of the 5' 440 bp of ground, indicating that transcription of that sequence was unde- F V n i INS (vector L X H S N ) revealed R N A steady-state levels tectable (Fig. S A , B ) . The vector sequences downstream of as low as those observed for L B H S N , 30-fold reduced comC F T R , represented by the S V 4 0 probe, yielded very low sig- pared to L X S N (Fig. 6 and Table 1). A deletion of 194 bp of nals compared to the background from NIH-3T3 cells (not sequence at the 3' end of the X H fragment to produce L X S S N shown). These results are consistent with a block to transcrip- resulted in R N A steady-state levels that were nine-fold reduced compared to L X S N (Table 1). The smaller fragment in the tional elongation near the middle of the C F T R c D N A . vector L X P S N , 392 bp in length, had a residual six-fold reduction in R N A steady-state levels. B y contrast, the smaller fragments in L P S S N (Fig. 6), L S H S N , and L B M S N had no signifDeletion analyses of the FVIII I N S fragment reveals icant inhibitory effect on vector expression (Table 1). Relative the presence of inhibitory sequence elements that R N A levels were normalized to the signal for a P-actin probe decrease expression in an additive m a n n e r that represented total R N A loaded in each lane. In summary, an Vectors containing smaller fragments derived from FVIU inhibitory sequence element in the fragment X P interacts with INS were found to have relatively high titers (Lynch et al., the downstream sequences within FVIII INS to downregulate 1993). T o allow the detection of possible interaction between steady-state R N A levels.
FACTOR VIII A N D CFTR TRANSCRIPTIONAL ELONGATION
475
LCFSN 1 0.9^ O.S-:0.7-;0.6-:0.5^0.4 0.34 0,2 0,1^Psi-LTR
Psi
CFl
FIG. 5. Nuclear mn-on analysis for L C F S N and untransduced N1H-3T3 cells. A. Graphs are similar to those in Fig. 3A. Each value represents signals from duplicate nuclear mn-on analyses performed simultaneously. B. A representative blot is shown for N1H-3T3 cells transduced with L C F S N and for untransduced NIH-3T3 cells. C. Probes derived from the C F T R c D N A are shown. The probes derived from the parent vector, L X S N , are shown in Fig. 3C. The insert for L C F S N was the C F T R cDNA.
CF2
B LCFSN
NIH 3T3
Psi- "^ LTR
CF1
PsiLTR
CF2
Psi
CF2
Psi
SV40
neo
H2B
neo
H2B
LIX .
actin
CFl ^ r
actin
CFl CFTR cDNA probes:
EcoRI
CF2
1 kb
The inhibitory effect of FVIII I N S w a s decreased when located downstream of neo Position dependence has been observed for the negative regulatory element ( N R E ) sequence from the bcl-2 gene, which had higher expression w h e n located further from the promoter in a position downstream of the neo gene (Young and Korsmeyer, 1993). A retroviral vector in which FVIII INS was located 1 k b downstream of the L T R promoter ( L N B H ) was constmcted by inserting the neo gene in a position upstream of F V U I I N S next to the L T R promoter. The level of full-length L N B H vector R N A level was elevated six-fold relative to the steady-state R N A level for the vector wifli F V I U I N S adjacent to the L T R ( L B H S N ) (Fig. 2). The signal for L N B H vector R N A was the same with a neo or F V U I INS probe, indicating tfiat the FVIII I N S D N A had not been deleted. The increase in steady-state R N A forflieL N B H vector compared to L B H S N demonstrates position dependence for FVIII INS.
later. Phosphorimager quantitation revealed that F V I U INS transcripts were induced approximately six-fold, similar to the extent that the FIX and neo transcripts were increased (Fig. 7) Thus, translation of a F V U I INS R N A does not appear to cause the inhibitory effect of F V I U INS in retroviral vectors.
DISCUSSION Transcriptional elongation is a control point for expression of the FVIII and C F T R c D N A s
The regulation of transcriptional elongation has been shown to be important to the control of expression of the c-myc and hsp-70 genes, and the T A R element of H I V ( K m m m et al., 1993). These blocks to transcriptional elongation have been shown to function in a promoter-proximal position, in contrast to F V U I I N S and the C F T R c D N A , which affect transcriptional elongation further downstream. The finding of decreased expression of the F V I U and C F T R coding regions on the basis of blocks to transcriptional elongation in vitro suggests that the T h e inhibitory effect of FVIII I N S w a s unaffected by control of transcriptional elongation might be important in detranslation termining the extremely low expression of these sequences in To evaluate the possible role of translation in decreased ex- vivo (Wion et al., 1985; Riordan et al., 1989). The F V I U I N S pression of F V i n INS, a cycloheximide translational arrest was R N A is as stable as the FIX vector R N A and neo R N A in an performed. N I H - 3 T 3 cells transduced with L B H S N or L I X S N assay of R N A degradation, which conoborates the relevance of were exposed to cycloheximide (10 p.g/ml) in the culture m e - the control of transcriptional elongation in the down-regulation dium, and total R N A was harvested at time points up to 5 hr of FVIII I N S expression. The potential role of R N A degrada-
476
K O E B E R L ET AL.
FVlll INS (BH) Inserts Restriction site: Bg/ll SamHI BsfXI D2
_L.
_L _L
Psfl
Sful I
H/ncll
Vector: LXSSN LXPSN LXHSN LD2SN LPSSN LSHSN LBMSN • FVIII INS deletio n cor structs
z CO 3
z C O Q . X -J
?: c /O 3 C X _l
z t o C O X —I
z X X
z C O 5)
z C O C CO L
z C O X _1 — Vector
• beta-actin
FIG. 6. Deletion analysis of F V U I INS. The indicated fragments of FVlll INS were inserted into the p L X S N retroviral vector. L D 2 S N consists of the X H fragment with a further 5' deletion of approximately 200 bp. Relative steady-state R N A levels, as determined by the neo signal from the Northem blot for various transcripts from the long terminal repeat (LTR) promoter and normalized to a p-actin signal, are listed in Table 1. Intermediate bands between the L T R transcripts and S V 4 0 neo transcripts presumably represent reananged provimses from pooled P A 3 1 7 packaging cell lines, as they were much reduced in N1H-3T3 cells transduced by a cloned P A 3 1 7 line producing L B H S N (Fig. 1). A n autoradiogram for the same blot hybridized to a chicken p-actin probe is shown to demonstrate approximately equivalent loading of each lane.
Table 1. Reduction is Steady-State RNA Levels for Vectors Containing Fragments of FVIU INS, Compared TO LXSN Vector
Fold reduction in R N A level
LBHSN LXHSN LD2SN LXSSN LXPSN LPSSN LBMSN LSHSN LXSN
33-100 33 25 9 6 1.5 Equivalent to LXSN Equivalent to LXSN Equivalent to LXSN
The relative vector R N A level for each vector, compared to LXSN, is shown. Vector R N A levels represent the signal for vector R N A from a Northern blot hybridized to a neo probe, normalized to the P-actin signal for that lane. Signals were quantified with a phosphorimager. The inhibitory effect of the F V H I INS exhibited position dependence, as evidenced by higher steady-state vector R N A levels when the inhibitory sequence was located 1 kb further from the promoter. F V U I INS expression for the L N B H vector, in which the neo gene was inserted upstream of F V U I INS, was increased six-fold compared to L B H S N , in which FVlll INS was 1 kb closer to the L T R promoter. A similar effect was demonstrated for the bcl-2 negative regulatory element ( N R E ) sequence, which is located in the 5' untranslated sequence of the bcl-2 gene and decreases transcription in mature B-cell lines (Young and Korsmeyer, 1993). Given the evidence for a block to transcriptional elongation in FVlll INS, the observed increase in steady-state R N A levels in both cases could be due to greater elongation competence by transcription complexes that have traversed the neo gene. The control of expression of the FVIII INS is not primarily regulated by R N A degradation, translation, R N A egress f r o m the nucleus, or splicing RNA degradation is a major regulatory mechanism for the expression of several mammalian genes, including c-myc and
tion in the control of C F T R c D N A expression has not been defined. The conelation of decreased steady-state R N A levels with blocks to transcriptional elongation within the FVlll INS and the C F T R c D N A indicates that transcriptional elongation is important to the control of expression in both cases. Earlier nuclear mn-on analysis of a plasmid FVlll expression vector in Chinese hamster ovary cells failed to detect a block to transcriptional elongation in the FVlll c D N A (Kaufman et al., 1989). The experimental design in this earlier work precluded detection of a block to transcriptional elongation within F V U I , because upstream probes that could have quantified a higher signal for transcriptional initiation, compared to the signal for the FVlll c D N A , were omitted (Kaufman et al., 1988, 1989). In addition, the promoter, vector, and cell type differed from those utilized here; thus, the results are not directly comparable. Although post-transcriptional control was suggested as the mechanism for decreased FVlll expression (Kaufman et al., 1990), our findings indicate that neither nuclear egress, R N A stability, nor translation are responsible for inhibition of F V U I INS expression.
LBHSN 0 5
LIXSN 0 5 hr
Vector
FIG. 7. Translational anest with cycloheximide. NIH-3T3 cells transduced with L B H S N or L I X S N were incubated with cycloheximide (10 p-g/ml) in the medium for 5 hr before total R N A was extracted. Ethidium bromide staining of ribosomal R N A indicated similar loading of each lane (not shown). The Northem blot was hybridized to a neo probe.
F A C T O R VIII A N D C F T R T R A N S C R I P T I O N A L E L O N G A T I O N
477
c-fos (Linial et al., 1985; Shyu et al, 1989; Wisdom and Lee, this sequence gradually decreased as progressive deletions were 1991). Transcriptional anest with actinomycin D induces the made. T w o sequence elements within FVIU INS (XP and PS) prompt disappearance of these transcripts, as demonstrated here had a synergistic inhibitory effect on expression from a retrovifor c-myc. Inhibitors of translation induce R N A levels for genes ral vector. The presence of multiple inhibitory elements that such as c-myc, P-tubulin, histone, and the transferrin receptor, interact to decrease the efficiency of transcriptional elongation where translation appears to be linked to R N A degradation is unique to FVUI, in contrast to the c-myc, hsp-70, and A D A (Greenberg and Ziff, 1984; Linial et al, 1985; Graves et al, genes and HIV T A R element, which contain a single block to 1987; Pachter et al, 1987; Mullner and Kuhn, 1988). For transcriptional elongation (Bentley and Groudine, 1986; example, a superinduction of 12- to 30-fold has been observed Kaufman et al, 1989; Ramamurthy et al, 1990; Bengal and for the c-myc m R N A in the presence of cycloheximide, and has Aloni, 1991; Lee etal, 1992; Kash era/., 1993). The presence been attributed to decreased R N A degradation (Linial et al, of a stem-loop stmcture has been conelated with a block to 1985; Wisdom and Lee, 1991). FVIU INS vector R N A per- transcriptional elongation in the HIV T A R element (Bengal and sisted for 6 hr in the presence of actinomycin D and was induced Aloni, 1991), but no stem-loop stmctures of similar size are only six-fold in the presence of cycloheximide, similar to the found in FVUI INS. FVIU sequences upstream of FVUI INS effects observed for the FIX vector R N A . These results indicate also appear to have an inhibitory effect on the expression of that R N A degradation is not a major cause of low expression for larger fragments of the c D N A (Lyncher a/., 1993). FVIU INS. W e have examined the FVlll INS element for sequence eleLow expression of FVIII and CFTR complicates efforts ments that have been shown to mediate R N A instability in other toward h u m a n gene therapy systems. Sequence elements that might suggest a role for R N A degradation are present in FVIU INS. Regions of high A T The advantage of retroviral vectors for delivery of CFTR and content in an inhibitory sequence from the gag gene of the FVlll might be to allow long-term expression of these c D N A s , human immunodeficiency vims (HIV), promote R N A degrada- which has not been possible with nonintegrating vectors tion in the absence of Rev protein, (Schwartz et al., 1992 a, b). ( D m m m et al, 1990; Rosenfeld et al, 1992). The use of Although small regions of high A T content are found in FVlll retroviral vectors for delivery of the FVIII and C F T R c D N A s is INS (Gitschier et al., 1984), they do not generate R N A instabil- complicated by the inherent low levels of transcription of these ity in this context. While destabilizing sequence elements found sequences. Retroviral vectors are dependent upon transcription in 3' untranslated regions of the cytokine genes are present in to produce vector R N A , and the low titers of FVIU and C F T R F V U I INS, these sequences are not sunounded by AT-rich vectors reflect inefficient transcription. Another disadvantage areas typical of destabilizing elements (Shaw and Kamen, of the FVlll vector is the need to transduce an extremely large 1986; Reeves et al, 1987). The presence of rare codons has number of cells to have a therapeutic effect, due to the low been conelated with R N A instability in the M A T alpha-1 gene production of F V U I protein from transduced cells (Kaufman et of Saccharomyces cerevisiae (Ca^nigTO et al., 1993). Several al, 1989; Lynch et al, 1993). A n advantage of integrating codons with frequencies less than 5 % among primates (Wada et vectors, such as retroviral vectors, for gene therapy is that al, 1992) are found in the FVIU inhibitory sequence, but the descendants of transduced cells are more likely to retain the distribution of these codons does not conespond to the location vector. Therefore, a retroviral FVIII expression vector with a hightiterand high FVlll expression would be useful for gene of smaller inhibitory fragments within FVIU INS. The correlation between relative levels of nuclear and cyto- therapy in hemophilia A. T o achieve this goal, the block to plasmic R N A for vectors with or without FVIII INS indicates transcriptional elongation in the FVlll c D N A would have to be that nuclear translocation and splicing do not mediate low F V U I inactivated. Although in vitro mutagenesis should allow this, INS expression. The Rev protein is critical to the translocation tiie diffuse nature of the FVIU inhibitory sequence will compliof H I V transcripts to the cytoplasm (Emerman et al., 1989), but cate the task. If the lOO-fold inhibition of the F V U I c D N A on F V U I INS vector R N A is not down-regulated at the step of vector R N A expression could be reduced to a lO-fold inhibinuclear egress. The relative steady-state levels of FVlll INS tion, the number of transduced cells needed to ameliorate seand L X S N vector R N A s are consistent in cytoplasmic, total, vere hemophilia to a mild course would be reduced to a more and nuclear R N A , indicating that low levels of FVIII INS R N A feasible SO million (Lynch et al, 1993). Our approach to this are determined by a nuclear process. Spliced FVIII vector problem will be to introduce nucleotide substitutions in the R N A s were not observed, and would not be expected, given the inhibitory regions of the F V U I c D N A , to inactivate these elelack of splice acceptor sites in the F V U I c D N A . Transcripts ments and to allow higher levels of FVIU production from intermediate in size between the full-length vector R N A and retroviral vectors. neo R N A (transcribed from the intemal S V 4 0 promoter) were seen with pooled populations of PA317 producers; apparentiy ACKNOWLEDGMENTS these smaller R N A species represent reananged proviral species that are frequently observed for the FVIU vectors (Lynch et We thank Mark Groudine for advice and suggestions during al, 1993). the course of these experiments and for providing nuclear mn-on facilities. W e thank John Olsen for the C F T R vector and Multiple sequence elements inhibit FVIII INS C F T R vector-producing packaging cell lines. This work was expression supported by the Judith Graham Pool Fellowship from the NaWe demonstrated that a single inhibitory element could nottional Hemophilia Foundation (D.D.K.), the Cystic Fibrosis be identified within FVIII INS, because the inhibitory effect of Foundation (C.L.H. and A.D.M.), a Special Fellowship from
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Postttanscriptional gene regulation and the Leukemia Society of America (A.K.), a grant from Targeted Genetics Corporation (A.D.M.), and grants HL412I2 and D K 4 7 7 5 4 from the National Instimtes of Health (A.D.M.).
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This article has been cited by: 1. J M Johnston, G Denning, C B Doering, H T Spencer. 2013. Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A. Gene Therapy 20:6, 607-615. [CrossRef] 2. S. W. Pipe, H. Miao, S. P. Butler, J. Calcaterra, W. H. Velander. 2011. Functional Factor VIII Made with Von willebrand Factor at High Levels in Transgenic Milk. Journal of Thrombosis and Haemostasis no-no. [CrossRef] 3. N. J. Ward, S. M. K. Buckley, S. N. Waddington, T. VandenDriessche, M. K. L. Chuah, A. C. Nathwani, J. McIntosh, E. G. D. Tuddenham, C. Kinnon, A. J. Thrasher, J. H. McVey. 2011. Codon optimization of human factor VIII cDNAs leads to high-level expression. Blood 117:3, 798-807. [CrossRef] 4. Christopher B Doering, H Trent Spencer. 2009. Advancements in gene transfer-based therapy for hemophilia A. Expert Review of Hematology 2:6, 673-683. [CrossRef] 5. P A Radcliffe, C J M Sion, F J Wilkes, E J Custard, G L Beard, S M Kingsman, K A Mitrophanous. 2008. Analysis of factor VIII mediated suppression of lentiviral vector titres. Gene Therapy 15:4, 289-297. [CrossRef] 6. Andrew Gómez-Vargas, Gonzalo HortelanoTransplants for hemophilia 187-203. [CrossRef] 7. Assem G Ziady, Pamela B Davis, Michael W Konstan. 2003. Non-viral gene transfer therapy for cystic fibrosis. Expert Opinion on Biological Therapy 3:3, 449-458. [CrossRef] 8. A. Van Damme, M. K. L. Chuah, F. Dell'accio, C. De Bari, F. Luyten, D. Collen, T. VandenDriessche. 2003. Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long-terminal repeats. Haemophilia 9:1, 94-103. [CrossRef] 9. T. Tonn, C. Herder, S. Becker, E. Seifried, M. Grez. 2002. Generation and Characterization of Human Hematopoietic Cell Lines Expressing Factor VIII. Journal of Hematotherapy & Stem Cell Research 11:4, 695-704. [Abstract] [Full Text PDF] [Full Text PDF with Links] 10. Carmen Garc�a-Mart�n, Marinee K. L. Chuah, An Van Damme, Kelly E. Robinson, Beatrijs Vanzieleghem, JeanMarie Saint-Remy, Dominique Gallardo, Frederick A. Ofosu, Thierry Vandendriessche, Gonzalo Hortelano. 2002. Therapeutic levels of human Factor VIII in mice implanted with encapsulated cells: potential for gene therapy of haemophilia A. The Journal of Gene Medicine 4:2, 215-223. [CrossRef] 11. K. John Pasi. 2001. Gene therapy for haemophilia. British Journal of Haematology 115:4, 744-757. [CrossRef] 12. Marinee K. L. Chuah, Desire Collen, Thierry VandenDriessche. 2001. Gene therapy for hemophilia. The Journal of Gene Medicine 3:1, 3-20. [CrossRef] 13. G. Emilien, J.-M. Maloteaux, C. Penasse, A. Goodeve, C. Casimir. 2000. Haemophilias: advances towards genetic engineering replacement therapy. Clinical & Laboratory Haematology 22:6, 313-323. [CrossRef] 14. Gonzalo Hortelano, Frederick A Ofosu. 2000. Therapeutic approaches for haemophilia. Expert Opinion on Therapeutic Patents 10:6, 929-938. [CrossRef] 15. Marinee K.L. Chuah, An Van Damme, Hans Zwinnen, Inge Goovaerts, Veerle Vanslembrouck, Desire Collen, Thierry Vandendriessche. 2000. Long-Term Persistence of Human Bone Marrow Stromal Cells Transduced with Factor VIII-Retroviral Vectors and Transient Production of Therapeutic Levels of Human Factor VIII in Nonmyeloablated Immunodeficient Mice. Human Gene Therapy 11:5, 729-738. [Abstract] [Full Text PDF] [Full Text PDF with Links] 16. Randal J. Kaufman. 1999. Advances toward Gene Therapy for Hemophilia at the Millennium. Human Gene Therapy 10:13, 2091-2107. [Abstract] [Full Text PDF] [Full Text PDF with Links] 17. Angela M. Gallo-Penn, Pamela S. Shirley, Julie L. Andrews, Dawn B. Kayda, Anne M. Pinkstaff, Michele Kaloss, Shawn Tinlin, Cherie Cameron, Colleen Notley, Christine Hough, David Lillicrap, Michael Kaleko, Sheila Connelly. 1999. In Vivo Evaluation of an Adenoviral Vector Encoding Canine Factor VIII: High-Level, Sustained Expression in Hemophiliac Mice. Human Gene Therapy 10:11, 1791-1802. [Abstract] [Full Text PDF] [Full Text PDF with Links] 18. C CHEN, X FANG, J ZHU, X WU, Z ZHANG, J GU, Z WANG, C CHI. 1999. The Gene Expression of Coagulation Factor VIII in Mammalian Cell Lines. Thrombosis Research 95:2, 105-115. [CrossRef] 19. C. A. Lee, C. M. Kessler, D. Varon, U. Martinowitz, M. Heim, S. CONNELLY, M. KALEKO. 1998. Haemophilia A gene therapy. Haemophilia 4:4, 380-388. [CrossRef] 20. Marinee K. L. Chuah, Hilde Brems, Veerle Vanslembrouck, Desire Collen, Thierry VandenDriessche. 1998. Bone Marrow Stromal Cells as Targets for Gene Therapy of Hemophilia A. Human Gene Therapy 9:3, 353-365. [Abstract] [Full Text PDF] [Full Text PDF with Links]
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