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Journal of Virology, October 1998, p. 8150-8157, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Development of a Self-Inactivating Lentivirus Vector

Hiroyuki Miyoshi, Ulrike Blömer,dagger Masayo Takahashi,Dagger Fred H. Gage, and Inder M. Verma*

Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037

Received 30 April 1998/Accepted 13 July 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have constructed a new series of lentivirus vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing cells. The U3 region of the 5' long terminal repeat (LTR) in vector constructs was replaced with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription but still maintaining high levels of expression. A self-inactivating (SIN) vector was constructed by deleting 133 bp in the U3 region of the 3' LTR, including the TATA box and binding sites for transcription factors Sp1 and NF-kappa B. The deletion is transferred to the 5' LTR after reverse transcription and integration in infected cells, resulting in the transcriptional inactivation of the LTR in the proviruses. SIN viruses can be generated with no significant decreases in titer. Injection of viruses into the rat brain showed that a SIN vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type vector. Interestingly, a wild-type vector without an internal promoter also successfully transduced neurons in the brain, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even in the absence of Tat. Furthermore, injection of viruses into the subretinal space of the rat eye showed that wild-type vector transduced predominantly retinal pigment epithelium and photoreceptor cells, while SIN vector was able to transduce other types of retinal cells, including bipolar, Müller, horizontal, and amacrine cells. This finding suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. SIN HIV vectors should be safer for gene therapy, and they also have broader applicability as a means of high-level gene transfer and expression in nondividing cells.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Gene therapy approaches rely on efficient transfer of genes to the desired target cells (for reviews, see references 12, 32, 34, 35, and 48). A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, retrovirus vectors derived from oncoretroviruses such as murine leukemia virus (MLV) have been the most widely used for gene therapy applications. However, a major problem with these retrovirus vectors is the requirement for proliferation of the target cells for integration, limiting their use for gene transfer into nondividing cells such as hepatocytes, myoblasts, neurons, and hematopoietic stem cells. In contrast, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells (7, 30, 49).

We have recently developed a lentivirus vector based on HIV-1 that can transduce nondividing cells in vitro and in vivo (38). These HIV vectors are pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G); hence they can transduce a broad range of tissues and can be concentrated to high titers. We have shown that HIV vectors can stably integrate into the host cell genome and obtained long-term expression of transgenes in brain, liver, muscle, and retina (4, 24, 33, 38, 39). No cellular immune response can be detected at the site of injection. Furthermore, second injection of the HIV vector into the animals is possible, indicating the lack of any potent humoral immune response to the vector (24).

Although HIV vectors promise great utility for gene therapy, there is concern about their safety since HIV-1 is the etiologic agent of AIDS. The major safety concern is the generation of replication-competent virus during the production of vectors. In this regard, we have minimized the possibility for generating replication-competent virus through recombination by using a three-plasmid expression system which consists of packaging, envelope, and vector constructs (38, 39). Furthermore, recent studies have demonstrated the possibility of eliminating all accessory genes (vif, vpr, vpu, and nef) from a packaging construct without losing the ability to transduce nondividing cells (24, 26, 43, 56).

Another safety concern about HIV vectors, as for MLV-based vectors, is the possibility of insertional activation of cellular oncogenes by random integration of the vector provirus into the host genome. To overcome this problem, we have constructed a self-inactivating (SIN) vector in which the viral enhancer and promoter sequences have been deleted. In this report, we show that SIN vectors can be generated and transduce nondividing cells in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilization by replication-competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR. A further modification has been made in the vector construct in which the U3 region of the 5' LTR has been replaced with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription with no decreases in viral titer. SIN vectors combined with this hybrid 5' LTR further reduce the possibility of recombination to generate replication-competent virus because there is no complete U3 sequence in the virus production system. These modifications add additional safety features to the HIV-1-based lentivirus vector system.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid constructions and preparation of viral stocks. Plasmid pHR'-MCS was constructed by replacing a 420-bp BamHI-XhoI fragment of pHR' (38) with a fragment of multiple cloning sites containing BamHI, Eco47III, BstXI, SacII, PstI, EcoRI, ClaI, XbaI, HpaI, and XhoI sites. To restructure the HIV-1 LTR and generate the SIN LTR, PCR amplification products of pHR'-MCS, an MluI-BspEI (-454 to -145) and a BspEI-ApaI (-144 to +180) fragment or an MluI-BspEI (-454 to -145) and a BspEI-ApaI (-8 to +180) fragment, were cloned into pcDNA3.1/Zeo(+) (Invitrogen) between the MluI and ApaI sites to generate pHIV-LTR or pHIV-SIN, respectively (numbers in parentheses indicate positions relative to the transcriptional start site). To construct HIV vectors pLL and pLS, a 2.3-kb BspEI fragment of pHR'-MCS was cloned into the BspEI sites of pHIV-LTR and pHIV-SIN, respectively. pLS has a 133-bp deletion (-141 to -9) in the 3' LTR. pHIV-Delta U3L and pHIV-Delta U3S were constructed by replacing a MluI-KasI (-454 to +183) fragment of pLL and pLS, respectively, with a MluI-KasI PCR amplification product (-13 to +183) to delete the U3 region of the 5' LTR. To generate the hybrid 5' LTR with the CMV promoter, pCL and pCS were constructed by inserting a PvuI-SacI fragment containing the CMV promoter from pcDNA3.1/Zeo(+) into the PvuI and MluI sites of pHIV-Delta U3L and pHIV-Delta U3S, respectively. The hybrid 5' LTR maintains the 23-bp distance between the TATA box of CMV promoter and the transcription start site in HIV-1 LTR. pLL-CG, pLS-CG, pCL-CG, and pCS-CG were constructed by inserting a 1.3-kb BamHI-XhoI fragment of pKS-CMV-GFP (33) containing the CMV-green fluorescent protein (GFP) expression cassette into the same sites of pLL, pLS, pCL, and pCS, respectively. pLL-G, pCL-G, and pCS-G were constructed by deleting the CMV promoter from pLL-CG, pCL-CG, and pCS-CG, respectively.

The VSV-G-pseudotyped HIV vectors were generated by transient cotransfection of a vector construct (15 µg) with the VSV-G-expressing construct pMD.G (5 µg) and the packaging construct pCMVDelta R8.2 (10 µg) into 293T cells as previously described (39). High-titer stocks of HIV vectors were prepared by ultracentrifugation. The titers of viruses were determined by infection of 293T cells or HeLa-CD4-LTR-beta -gal cells (27), seeded in six-well plates at 105 cells/well the day before infection, with serial dilutions of the vector stocks. After overnight incubation, the cell culture medium was changed; 48 h later, the number of GFP-positive cells was scored to quantify the titer. The vector stocks were assayed for p24 antigen levels by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) kit (DuPont NEN). After concentration by ultracentrifugation, titers of 1 × 109 to 2 × 109 transducing units (TU)/ml were usually obtained. Absence of replication-competent virus was determined by the marker rescue assay and measuring p24 antigen level by ELISA as described elsewhere (39).

Southern and Northern blot analyses. Genomic DNA was isolated from cells infected with virus by the procedure of Wu et al. (51). DNA (10 µg) was digested with the restriction enzymes BspEI and BamHI, separated on a 0.7% agarose gel, and transferred to a Hybond-N+ membrane (Amersham) in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Total cellular RNA was isolated from virus-infected cells by using an RNAqueous phenol-free total-RNA isolation kit (Ambion). RNA (10 µg) was separated on a 1% agarose-formaldehyde gel and transferred to a Hybond-N+ membrane in 25 mM potassium phosphate (pH 6.5). Membranes were UV irradiated with a UV cross-linker (Hoefer), hybridized with 32P-labeled probes generated by a Megaprime DNA labeling kit (Amersham) in Rapid-hyb buffer (Amersham) for 2 h at 65°C, washed twice in 0.1× SSC-0.1% sodium dodecyl sulfate for 15 min at 65°C, and exposed to an X-Omat AR X-ray film (Kodak). The BN and GFP probes were isolated from pCL-CG as 0.43-kb BssHII-NotI and 0.75-kb Eco47III-XhoI fragments, respectively.

In vivo experiments. High-titer stocks of HIV vectors (1 × 109 to 2 × 109 TU/ml) were used to inject the brain and eye. The titers of CL-G and CS-G vectors were normalized for HIV-1 p24 antigen levels. Adult female Fischer 344 rats were anesthetized by intramuscular injection of ketamine-acepromazine-xylazine. Three microliters of vector stock (3 × 106 TU) was injected into the striatum and the hippocampus bilaterally (n = 4 for each vector) with a 5-µl Hamilton syringe. Rats were sacrificed at 2 and 6 weeks postinjection.

Fischer 344 rat pups (age 2 to 5 days) were anesthetized by chilling on ice for 5 min. The eyeball was exposed by an incision in the eyelid, parallel to the edge of the open eyelid. Subretinal injections were performed under an operating microscope. A 0.5-µl volume of vector stock (106 TU) was injected into the subretinal space (n = 8 for each vector), using a glass capillary connected with 5-µl Hamilton syringe. Rats were sacrificed at 6 and 12 weeks postinjection.

Rats were intracardially perfused with cold fixing solution (4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer). The tissues were removed, further fixed in the fixing solution, and cryoprotected in 30% sucrose at 4°C overnight. The tissues were then frozen in Tissue-Tek O.C.T. compound (Sakura Finetek) on dry ice and sectioned on a sliding microtome (50-µm-thick brain sections) or a cryostat (20-µm-thick eye sections). Immunofluorescence staining was performed as previously described (4). The sections were analyzed by confocal laser scanning microscopy (Bio-Rad). The signals were collected, digitally color enhanced, and superimposed.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction and generation of modified HIV vectors. In the life cycle of retroviruses, the U3 region of the 3' LTR is duplicated to form the corresponding region of the 5' LTR during the process of reverse transcription and viral DNA synthesis in infected cells. This mechanism of viral replication allowed us to make two principal modifications to the HIV vector, as shown in Fig. 1. First, the U3 region of the 5' LTR was replaced with the CMV promoter. Second, 133 bp in the U3 region of the 3' LTR, which contains the TATA box and binding sites for transcription factors Sp1 and NF-kappa B, were deleted. This deletion will be transferred to the 5' LTR after reverse transcription. Consequently, the transcriptional unit from the LTRs in a provirus is eliminated. This type of vector is called a SIN vector (54).


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  		FIG. 1.   Structures of HIV vector constructs and corresponding proviruses. (Left) HIV vector constructs. Each vector construct is cotransfected with the packaging and VSV-G expression constructs into 293T cells. Viral transcription initiates at the U3/R border in the 5' LTR and terminates at the R/U5 border in the 3' LTR. The viral RNA is packaged into virions. Virus is harvested and used to infect target cells. Triangles represent deleted U3 region. (Right) Structures of integrated proviruses. In infected cells, the U3 region of the 3' LTR is used as a template for the synthesis of the U3 region in both LTRs during the process of reverse transcription of the viral RNA into double-stranded DNA. As a result, the U3 region of the 3' LTR is duplicated and transferred to the 5' LTR in the integrated provirus. The BspEI-BamHI restriction fragments expected in Southern blot analysis (Fig. 2) and the RNA transcripts expected in Northern blot analysis (Fig. 3) are shown below each proviral structure with their sizes. The locations of the BN and GFP probes used in Southern blot and Northern blot analyses are also indicated.

A series of vectors termed LL-CG, LS-CG, CL-CG, and CS-CG (Fig. 1) were constructed to determine whether these modifications would alter the titer of virus generated from the vectors. All vectors contain the GFP gene as a reporter with an internal CMV promoter. CL-CG and CS-CG vectors contain the CMV promoter in place of the U3 region of the 5' LTR. The virus generated from CL-CG and CS-CG should contain the same RNA genome as the virus from LL-CG and LS-CG, respectively (Fig. 1). We also constructed CL-G and CS-G vectors without an internal promoter, in which the GFP gene is under the control of the 5' LTR promoter.

VSV-G-pseudotyped vectors were generated by transient cotransfection of each vector construct with a VSV-G expression construct and a packaging construct into 293T cells. The vector virus was harvested 62 h after transfection and used to infect 293T cells to determine the viral titer by quantitation of the number of GFP-positive cells. The LL-CG vector yielded a mean titer of 6 × 105 TU/ml. As shown in Table 1, replacement of the U3 region of the 5' LTR with the CMV promoter did not reduce the viral titers. The titers of SIN vectors LS-CG and CS-CG were approximately the same as those of their wild-type counterparts, LL-CG and CL-CG, respectively. These results indicate that the small deletion in the U3 region in SIN vectors did not significantly affect titers. Lower titers of CL-G may reflect the relative inefficiency of the LTR promoter because of the absence of the trans activator Tat in the infected cells. A few cells were scored positive for GFP with the CS-G vector, probably due to integration events near host genome promoters since there is no promoter in the provirus. To measure viral production with CL-G and CS-G vectors, the levels of HIV-1 p24 antigen in the supernatants were measured, as these correlate with the amount of virions. The levels of p24 obtained with the CL-G and CS-G vectors were roughly equivalent to those obtained with other vectors, suggesting approximately similar levels of virus production with all vectors (Table 1). However, we cannot rule out the possibility of generating virions lacking viral genome RNA with this assay.

                              
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  		TABLE 1.   Titers of HIV vectors

Expression from the hybrid 5' LTR with the CMV promoter is Tat independent. It has been shown that Tat trans activation of the HIV-1 LTR requires not only the Tat-responsive region (TAR) in the R region of the LTR but also the TATA box and binding sites for Sp1 and NF-kappa B (2, 3, 17, 22, 31, 36, 41, 55). To verify that the hybrid CMV-LTR promoter is Tat independent, the promoter activity of the CL-G vector construct was compared with that of construct LL-G, in which the internal CMV promoter was deleted from LL-CG and therefore the GFP gene was expressed under the control of wild-type 5' LTR. These constructs were transfected into 293T or HeLa cells with either a Tat expression plasmid or a control plasmid, and levels of GFP in cell extracts were measured 48 h later by fluorescence spectroscopy (40). Table 2 shows that the GFP expression in cells transfected with LL-G was increased three- to sixfold in the presence of Tat. The low level of trans activation by Tat in 293T cells is probably caused by the higher level of expression from the LTR due to trans activation by the adenovirus E1A gene product expressed in 293T cells (37). On the other hand, CL-G was not responsive to Tat, though levels of expression were similar to those obtained with LL-G in the presence of Tat. Thus, the replacement of U3 region of the 5' LTR with the CMV promoter resulted in loss of Tat responsiveness, but the basal activity was comparable to that of wild-type LTR in the presence of Tat in 293T and HeLa cells.

                              
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  		TABLE 2.   Transcriptional activities of LL-G and CL-G constructsa

Characterization of proviral structure and transcription. The structures of proviruses were characterized by Southern blot analysis. HeLa-CD4-LTR-beta -gal cells (27) were infected with each vector and cultured for 36 days with 11 passages. Genomic DNA was isolated from infected cells, digested with the restriction enzymes BspEI and BamHI, and hybridized with a BN probe (Fig. 2). The predicted structures of proviruses are shown in Fig. 1. BspEI cleaves once in each LTR, immediately upstream of the deleted region in SIN vectors, and BamHI cleaves once downstream of the 5' LTR. Therefore, a 1.69-kb BspEI-BamHI fragment should be generated from provirus containing wild-type 5' LTR when hybridized with the BN probe. If the 133-bp deletion present in the 3' LTR of SIN vector is transferred to the 5' LTR of the integrated provirus, a 1.56-kb BspEI-BamHI fragment should be generated in place of the 1.69-kb fragment. As shown in Fig. 2, the expected fragments were generated from each provirus. Analysis of infected 293T cells yielded identical results (data not shown).


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  		FIG. 2.   Southern blot analysis of the integrated proviral structure. Genomic DNA isolated from HeLa-CD4-LTR-beta -gal cells infected with HIV vectors was digested with BspEI and BamHI. The blot was hybridized with the BN probe. The vector used for infection is indicated above each lane. Control, uninfected HeLa-CD4-LTR-beta -gal cells. The expected sizes of fragments are shown in Fig. 1. Size markers are indicated on the left.

We next analyzed the transcriptional activities of proviruses. Total cellular RNA was isolated from HeLa-CD4-LTR-beta -gal cells infected with each virus described above, and Northern blot analysis was performed with the GFP gene as a probe. As shown in Fig. 3, expression of the expected transcripts depicted in Fig. 1 was observed for each of the proviruses. In the provirus derived from the LL-CG or CL-CG vector, three transcripts were produced: two viral transcripts initiated in the 5' LTR, a 3.60-kb full-length form and a 2.44-kb spliced form; and a 1.48-kb transcript initiated in the internal CMV promoter. All transcripts terminated at the polyadenylation signal in the R region of the 3' LTR. The full-length and spliced forms were confirmed by using the BN probe, which detects only the full-length form (data not shown; see Fig. 1 for structures). The level of transcripts expressed from the internal CMV promoter is higher than that of transcripts initiated in the 5' LTR. In the case of SIN vectors LS-CG and CS-CG, transcripts expressed from the 5' LTR were undetectable and a 1.35-kb transcript expressed from the internal promoter was detected. This transcript is shorter than the corresponding transcript from the LL-CG or CL-CG provirus because of the 133-bp deletion in the U3 region of the 3' LTR. The provirus derived from the CL-G vector produced detectable levels of the full-length and spliced viral transcripts. As expected, no RNA transcripts were detected in the cells infected with the CS-G vector. The same results were obtained with 293T cells infected with each virus (data not shown).


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  		FIG. 3.   Northern blot analysis of the proviral expression. Total cellular RNA was isolated from HeLa-CD4-LTR-beta -gal cells infected with HIV vectors. The blot was hybridized with the GFP probe and rehybridized with the human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe (Clontech). The vector used for infection is indicated above each lane. Control, uninfected HeLa-CD4-LTR-beta -gal cells. The expected size of each transcript is shown in Fig. 1. Size markers are indicated on the left.

Rescue of virus from cells infected with SIN vectors. To determine if there was any transcriptional activity from the 5' LTR of the SIN provirus, we used a more sensitive virus rescue assay. The VSV-G-expressing construct and the packaging construct were transfected into 293T cells previously infected with SIN vectors. If RNA was initiated in the 5' LTR of the SIN provirus, it would be packaged into viral particles and released into the medium. Such virus would then be detected by infection of virgin 293T cells. The results showed that 3 to 8 TU of virus per ml was rescued from cells previously infected with the LS-CG or CS-CG vector, whereas 5 × 104 to 8 × 104 TU/ml was rescued from cells infected with the LL-CG or CL-CG vector (Table 3). It is unlikely that the rescued virus arose through recombination during transfection and regenerated a functional 5' LTR because there is no complete U3 sequence, especially in the case of CS-CG. In fact, the virus preparations used for infection were shown to be free of replication-competent virus by the marker rescue assay (39). Viruses were able to be rescued presumably because SIN vectors integrated near an active cellular promoter and expressed the proviral genome, as seen with the CS-G vector (Table 1). On the basis of Northern blot analysis and virus rescue, it is apparent that effective transcription from the 5' LTR of the SIN provirus was inactivated.

                              
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  		TABLE 3.   Titers of viruses rescued by transient transfection of packaging and VSV-G constructsa

In vivo delivery of modified vectors. To transduce terminally differentiated neurons in vivo, high-titer stocks (1 × 109 to 2 × 109 TU/ml) of the CS-CG and CL-CG vectors were injected into the striatum and hippocampus of adult rat brains. The CL-G and CS-G vectors, normalized for equal amounts of p24 antigen, were also injected to detect the expression from the 5' LTR of wild-type and SIN vectors. At 2 and 6 weeks postinjection, the rats were sacrificed and the brains were cryosectioned and analyzed for GFP expression by fluorescence microscopy. As shown in Fig. 4, the CS-CG vector was found to transduce at efficiencies comparable to those of the CL-CG vector. Interestingly, the CL-G vector gave similar transduction efficiencies as well. This observation is consistent with previous studies showing high levels of expression from the HIV-1 LTR through constitutively active NF-kappa B in neurons (10, 25, 42). In brains injected with the CS-G vector, very few GFP-positive cells could be detected, and only at higher magnification. This was most likely due to proviral integration near a cellular promoter and expression of GFP, as seen in infection in vitro. There were no significant differences in the GFP transduction frequencies of the vectors between 2 and 6 weeks postinjection (data not shown). The nature of transduced cells was determined by immunofluorescence staining with three markers: NeuN for neurons, glial fibrillary acidic protein for astrocytes, and RIP for oligodendrocytes. The results showed that the majority of cells transduced with each vector were terminally differentiated neurons expressing NeuN, consistent with our previous observation (4, 38, 39). Additional immunofluorescence staining showed that cells transduced with the CS-CG vector were frequently labeled with choline acetyltransferase, which is a marker of cholinergic neurons (data not shown). In contrast, the CL-CG vector-transduced cells were rarely labeled with this marker.


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  		FIG. 4.   Expression of GFP in the striatum and hippocampus of adult rat brains 6 weeks after injection of HIV vectors. Sections were counterstained with propidium iodide. The vectors used for injection are indicated on the left. Inset, higher magnification of a GFP-positive cell.

The CL-CG and CS-CG vectors were also injected into the subretinal space of rat eyes, and transduction was evaluated 6 and 12 weeks later. Retinal cell types can be readily identified by their positions in the different retinal layers and by their distinctive morphologies. In the eyes injected with the wild-type vector CL-CG, GFP was predominantly expressed in retinal pigment epithelium and photoreceptor cells (Fig. 5A). On the other hand, the SIN vector CS-CG efficiently transduced not only retinal pigment epithelium and photoreceptor cells but also other retinal cells in inner nuclear layer, including bipolar, Müller, horizontal, and amacrine cells (Fig. 5B). This result together with that for the brain suggests that transcription from the HIV-1 LTR may have inhibitory effects on transcription from the internal promoter in some cell types. There were no significant differences in the patterns and transduction frequencies of the vectors between 6 and 12 weeks postinjection (data not shown).


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  		FIG. 5.   Expression of GFP in the retina of rat pups 12 weeks after injection of the CL-CG vector (A) and the CS-CG vector (B). Inset, higher magnification of GFP-positive inner nuclear layer (INL) cells. Scale bars, 10 µm. RPE, retinal pigment epithelium; ONL, outer nuclear layer.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

HIV-1, unlike other retroviruses such as MLV, is characterized by a complex genome that encodes two regulatory proteins (Tat and Rev) and four accessory proteins (Vif, Vpr, Vpu, and Nef) in addition to the common gag, pol, and env gene products. One of the accessory proteins, Tat, is an essential nuclear protein that augments levels of viral RNA by increasing transcriptional initiation and/or elongation (reviewed in reference 23). To manifest its function, Tat binds to a nascent RNA stem-loop structure of TAR, located at the 5' end of all viral transcripts. Tat trans activation has also been reported to require the TATA box and additional sequences in the 5' LTR, including binding sites for Sp1 and NF-kappa B (2, 3, 17, 22, 31, 36, 41, 55). This is consistent with our result that replacement of the U3 region of the 5' LTR with the CMV promoter resulted in Tat-independent transcription (Table 2). In the absence of Tat, this hybrid CMV-LTR promoter can drive high levels of expression comparable to those of the wild-type LTR in the presence of Tat. This allows production of HIV vectors in a system devoid of Tat. Indeed, Kim et al. (26) have recently reported that an HIV vector containing a similar promoter swap could generate virus with no significant decrease in titer in the absence of Tat. Recent studies have demonstrated the possibility of eliminating all accessory genes from a packaging construct without losing the ability to transduce nondividing cells (24, 26, 43, 56), although some accessory proteins appeared to be required for maximum transduction of specific tissues (e.g., Vpr for macrophages and Vpr and/or Vif for liver) (24, 56). Taken together, these observations suggest the possibility of eliminating all regulatory and accessory genes except rev from the HIV vector production system. Rev, together with the Rev response element in the vector, is required for efficient export of full-length vector transcripts to the cytoplasm. However, it may be possible to substitute Rev function by replacement with a protein serving a similar function (28).

The transcription of HIV-1 is directed by regulatory sequences in the 5' LTR. The core element in the U3 region, containing a canonical TATA box and three Sp1 binding sites, is essential for basal promoter activity and viral replication (2, 3, 17, 22, 41, 55). Immediately upstream of the Sp1 binding sites, tandem binding sites for NF-kappa B constitute an activation-dependent enhancer element (31, 36). Elimination of all Sp1 and NF-kappa B binding sites results in essentially total inactivation of viral replication (29, 44). Although a number of enhancer elements that modulate transcriptional activity have been identified upstream of the NF-kappa B binding sites, none of them can compensate for the loss of Sp1 and NF-kappa B binding sites. In the SIN HIV vector described in this study, as expected, the deletion of the TATA box and binding sites for Sp1 and NF-kappa B resulted in transcriptional inactivation of the LTR in the provirus in infected cells in vitro and in vivo. A notable aspect of the expression from the HIV-1 LTR is its low basal activity in the absence of Tat. In addition, Tat is not packaged into virions. Therefore, in most cells infected with HIV vectors, low levels of expression from the LTR would be expected because of the absence of Tat. However, we have demonstrated high-level expression from the LTR in the brain (Fig. 4), consistent with the finding that transgenic mice expressed an HIV-1 LTR-driven reporter gene at high levels in the brain (10). It has been reported that strong HIV-1 LTR promoter activity in neurons is mainly due to the presence of constitutively active NF-kappa B (25, 42). One safety concern about HIV vectors is the activation of proto-oncogenes through the LTR resulting from random integration of provirus into the host genome. In this regard, the SIN vector should reduce the potential of insertional activation. The transcriptional inactivation of the LTR in the SIN provirus should also prevent mobilization by replication-competent virus and minimize any risk of vector virus spread. It can also be argued that the rearranged viruses may arise through recombination during transfection and regenerate a wild-type LTR. Since SIN vectors combined with the hybrid CMV-LTR promoter (e.g., CS-CG) contain a single U3 region with a deletion, there is no complete U3 sequence in the virus production system: recombination to regenerate a wild-type U3 is not possible with such vectors. Furthermore, this combined modification minimizes the risk of recombination to generate replication-competent virus, although we have not detected replication-competent virus so far.

The application of MLV-based SIN vectors has been limited, since deletion of the TATA box resulted in low titers (9, 20, 21, 45, 53, 54). This is probably because the region of the TATA box in the 3' LTR plays an important role in processing of the 3' end of the viral RNA through secondary-structure interactions. Therefore, most SIN MLV vectors had a deletion only in the enhancer region, and transcriptional inactivation of the LTR was relatively inefficient (9, 20, 45, 54). In HIV-1, sequences within the U3 region, especially between the TATA box and the transcription initiation site, have been shown to be required for efficient 3' end processing (6, 8, 13, 18, 46, 47). These sequences direct the stable binding of cleavage and polyadenylation specificity factor, the factor responsible for recognition of the AAUAAA hexamer, to the poly(A) site and enhance the efficiency of 3'-end processing (19). However, Northern blot analysis of RNA from 293T cells transfected with the SIN vector construct showed no decreased levels of transcripts (data not shown) compared with the wild-type vector, consistent with no significant reduction in viral titer (Table 1). This finding suggests that the deletion of U3 sequences in the SIN vector may not affect the efficiency of 3'-end processing.

The results obtained from in vivo experiments revealed that the SIN vector can improve the expression of transgene in cholinergic neurons compared to the wild-type vector. Similarly, additional retinal cell types also showed high levels of transgene expression with the SIN vector (Fig. 5). It seems likely that the expression from the internal CMV promoter is negatively influenced by expression from the HIV-1 LTR in some cell types. This phenomenon, called transcriptional interference, has been reported for other retrovirus vectors (1, 5, 11, 14, 15, 45, 50, 52). Our results suggest that deletion of the U3 region of the LTR in HIV vectors may result in efficient expression from internal promoters in some tissues. Furthermore, SIN vectors should be particularly useful to introduce tissue-specific or regulatable promoters, since the LTR can have no cis-acting influence on these internal promoters. SIN HIV vectors should not only increase the safety of HIV vector-mediated gene therapy but also have general utility for high-efficiency transduction of genes into nondividing cells.

    ACKNOWLEDGMENTS

We are grateful to N. Somia and T. Kafri for critical reading of the manuscript. We thank members of the Verma and Gage laboratories for helpful suggestions.

H.M. was supported by a Uehara Memorial Foundation fellowship, and M.T. was supported by a Nippon Eye Bank Association fellowship. This work was supported by grants from the National Institutes of Health and Frances Berger Foundation. I.M.V. is an American Cancer Society Professor of Molecular Biology.

    FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Genetics, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92186-5800. Phone: (619) 453-4100, ext. 1462. Fax: (619) 558-7454. E-mail: verma{at}salk.edu.

dagger Present address: Department for Neurosurgery, Medical School Hannover, 30625 Hannover, Germany.

Dagger Present address: Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Armentano, D., S.-F. Yu, P. W. Kantoff, T. V. Ruden, W. F. Anderson, and E. Gilboa. 1987. Effect of internal viral sequences on the utility of retroviral vectors. J. Virol. 61:1647-1650[Abstract/Free Full Text].
2. Berkhout, B., A. Gatignol, A. B. Rabson, and K.-T. Jeang. 1990. TAR-independent activation of the HIV-1 LTR: evidence that tat requires specific regions of the promoter. Cell 62:757-767[Medline].
3. Berkhout, B., and K.-T. Jeang. 1992. Functional roles for the TATA promoter and enhancers in basal and tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat. J. Virol. 66:139-149[Abstract/Free Full Text].
4. Blömer, U., L. Naldini, T. Kafri, D. Trono, I. M. Verma, and F. H. Gage. 1997. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J. Virol. 71:6641-6649[Abstract].
5. Bowtell, D. D. L., S. Cory, G. R. Johnson, and T. J. Gonda. 1988. Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes. J. Virol. 62:2464-2473[Abstract/Free Full Text].
6. Brown, P. H., L. S. Tiley, and B. R. Cullen. 1991. Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences. J. Virol. 65:3340-3343[Abstract/Free Full Text].
7. Bukrinsky, M. I., N. Sharova, M. P. Dempsey, T. L. Stanwick, A. G. Bukrinskaya, S. Haggerty, and M. Stevenson. 1992. Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc. Natl. Acad. Sci. USA 89:6580-6584[Abstract/Free Full Text].
8. Cherrington, J., and D. Ganem. 1992. Regulation of polyadenylation in human immunodeficiency virus (HIV): contribution of promoter proximity and upstream sequences. EMBO J. 11:1513-1524[Medline].
9. Cone, R. D., A. Weber-Benarous, D. Baorto, and R. C. Mulligan. 1987. Regulated expression of a complete human beta -globin gene encoded by a transmissible retrovirus vector. Mol. Cell. Biol. 7:887-897[Abstract/Free Full Text].
10. Corboy, J. R., J. M. Buzy, M. C. Zink, and J. E. Clements. 1992. Expression directed from HIV long terminal repeats in the central nervous system of transgenic mice. Science 258:1804-1808[Abstract/Free Full Text].
11. Correll, P. H., S. Colilla, and S. Karlsson. 1994. Retroviral vector design for long-term expression in murine hematopoietic cells in vivo. Blood 84:1812-1822[Abstract/Free Full Text].
12. Crystal, R. G. 1995. Transfer of genes to humans: early lessons and obstacles to success. Science 270:404-410[Abstract/Free Full Text].
13. DeZazzo, J. D., J. E. Kilpatrick, and M. J. Imperiale. 1991. Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation. Mol. Cell. Biol. 11:1624-1630[Abstract/Free Full Text].
14. Emerman, M., and H. M. Temin. 1984. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39:459-467.
15. Emerman, M., and H. M. Temin. 1986. Quantitative analysis of gene suppression in integrated retrovirus vectors. Mol. Cell. Biol. 6:792-800[Abstract/Free Full Text].
16. Frankel, A. D., and C. O. Pabo. 1988. Cellular uptake of the Tat protein from human immunodeficiency virus. Cell 55:1189-1193[Medline].
17. Garcia, J. A., F. K. Wu, R. Mitsuyasu, and R. B. Gaynor. 1987. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus. EMBO J. 6:3761-3770[Medline].
18. Gilmartin, G. M., E. S. Fleming, and J. Oetjen. 1992. Activation of HIV-1 pre-mRNA 3' processing in vitro requires both an upstream element and TAR. EMBO J. 11:4419-4428[Medline].
19. Gilmartin, G. M., E. S. Fleming, J. Oetjen, and B. R. Graveley. 1995. CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition. Genes Dev. 9:72-83[Abstract/Free Full Text].
20. Guild, B. C., M. H. Finer, D. E. Housman, and R. C. Mulligan. 1988. Development of retrovirus vectors useful for expressing genes in cultured murine embryonal cells and hematopoietic cells in vivo. J. Virol. 62:3795-3801[Abstract/Free Full Text].
21. Hawley, R. G., L. Covarrubias, T. Hawley, and B. Mintz. 1987. Handicapped retroviral vectors efficiently transduce foreign genes into hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 84:2406-2410[Abstract/Free Full Text].
22. Jones, K. A., J. T. Kadonaga, P. A. Luciw, and R. Tjian. 1986. Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. Science 232:755-759[Abstract/Free Full Text].
23. Jones, K. A., and B. M. Peterlin. 1994. Control of RNA initiation and elongation at the HIV-1 promoter. Annu. Rev. Biochem. 63:717-743[Medline].
24. Kafri, T., U. Blömer, D. A. Peterson, F. H. Gage, and I. M. Verma. 1997. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat. Genet. 17:314-317[Medline].
25. Kaltschmidt, C., B. Kaltschmidt, H. Neumann, H. Wekerle, and P. A. Baeuerle. 1994. Constitutive NF-kappa B activity in neurons. Mol. Cell. Biol. 14:3981-3992[Abstract/Free Full Text].
26. Kim, V. N., K. Mitrophanous, S. M. Kingsman, and A. J. Kingsman. 1998. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J. Virol. 72:811-816[Abstract/Free Full Text].
27. Kimpton, J., and M. Emerman. 1992. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta -galactosidase gene. J. Virol. 66:2232-2239[Abstract/Free Full Text].
28. Krug, R. M. 1993. The regulation of export of mRNA from nucleus to cytoplasm. Curr. Opin. Cell Biol. 5:944-949[Medline].
29. Leonard, J., C. Parrott, A. J. Buckler-White, W. Turner, E. K. Ross, M. A. Martin, and A. B. Rabson. 1989. The NF-kappa B binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity. J. Virol. 63:4919-4924[Abstract/Free Full Text].
30. Lewis, P., M. Hensel, and M. Emerman. 1992. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J. 11:3053-3058[Medline].
31. Liu, J., N. D. Perkins, R. M. Schmid, and G. J. Nabel. 1992. Specific NF-kappa B subunits act in concert with tat to stimulate human immunodeficiency virus type 1 transcription. J. Virol. 66:3883-3887[Abstract/Free Full Text].
32. Miller, A. D. 1992. Human gene therapy comes of age. Nature 357:455-460[Medline].
33. Miyoshi, H., M. Takahashi, F. H. Gage, and I. M. Verma. 1997. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc. Natl. Acad. Sci. USA 94:10319-10323[Abstract/Free Full Text].
34. Morgan, R. A., and W. F. Anderson. 1993. Human gene therapy. Annu. Rev. Biochem. 62:191-217[Medline].
35. Mulligan, R. C. 1993. The basic science of gene therapy. Science 260:926-932[Abstract/Free Full Text].
36. Nable, G., and D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature 326:711-713[Medline].
37. Nabel, G. J., S. A. Rice, D. M. Knipe, and D. Baltimore. 1988. Alternative mechanisms for activation of human immunodeficiency virus enhancer in T cells. Science 239:1299-1302[Abstract/Free Full Text].
38. Naldini, L., U. Blömer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D. Trono. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267[Abstract].
39. Naldini, L., U. Blömer, F. H. Gage, D. Trono, and I. M. Verma. 1996. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93:11382-11388[Abstract/Free Full Text].
40. Niswender, K. D., S. M. Blackman, L. Rohde, M. A. Magnuson, and D. W. Piston. 1995. Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. J. Microsc. 180:109-116[Medline].
41. Olsen, H. S., and C. A. Rosen. 1992. Contribution of the TATA motif to tat-mediated transcriptional activation of human immunodeficiency virus gene expression. J. Virol. 66:5594-5597[Abstract/Free Full Text].
42. Rattner, A., M. Korner, M. D. Walker, and Y. Citri. 1993. NF-kappa B activates the HIV promoter in neurons. EMBO J. 12:4261-4267[Medline].
43. Reiser, J., G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert. 1996. Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. Proc. Natl. Acad. Sci. USA 93:15266-15271[Abstract/Free Full Text].
44. Ross, E. K., A. J. Buckler-White, A. B. Rabson, G. Englund, and M. A. Martin. 1991. Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell type. J. Virol. 65:4350-4358[Abstract/Free Full Text].
45. Soriano, P., G. Friedrich, and P. Lawinger. 1991. Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J. Virol. 65:2314-2319[Abstract/Free Full Text].
46. Valsamakis, A., S. Zeichner, S. Carswell, and J. C. Alwine. 1991. The human immunodeficiency virus type 1 polyadenylation signal: a 3' long terminal repeat element upstream of the AAUAAA necessary for efficient polyadenylation. Proc. Natl. Acad. Sci. USA 88:2108-2112[Abstract/Free Full Text].
47. Valsamakis, A., N. Schek, and J. C. Alwine. 1992. Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro. Mol. Cell. Biol. 12:3699-3705[Abstract/Free Full Text].
48. Verma, I. M., and N. Somia. 1997. Gene therapy---promises, problems and prospects. Nature 389:239-242[Medline].
49. Weinberg, J. B., T. J. Matthews, B. R. Cullen, and M. H. Malim. 1991. Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes. J. Exp. Med. 174:1477-1482[Abstract/Free Full Text].
50. Williams, D. A., S. H. Orkin, and R. C. Mulligan. 1986. Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo. Proc. Natl. Acad. Sci. USA 83:2566-2570[Abstract/Free Full Text].
51. Wu, Q., M. Chen, M. Buchwald, and R. A. Phillips. 1995. A simple, rapid method for isolation of high quality genomic DNA from animal tissues. Nucleic Acids Res. 23:5087-5088[Free Full Text].
52. Xu, L., J.-K. Yee, J. A. Wolff, and T. Friedmann. 1989. Factors affecting long-term stability of Moloney murine leukemia virus-based vectors. Virology 171:331-341[Medline].
53. Yee, J.-K., J. C. Moores, D. J. Jolly, J. A. Wolff, J. G. Respess, and T. Friedmann. 1987. Gene expression from transcriptionally disabled retroviral vectors. Proc. Natl. Acad. Sci. USA 84:5197-5201[Abstract/Free Full Text].
54. Yu, S.-F., T. V. Rüden, P. W. Kantoff, C. Garber, M. Seiberg, U. Rüther, W. F. Anderson, E. F. Wagner, and E. Gilboa. 1986. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl. Acad. Sci. USA 83:3194-3198[Abstract/Free Full Text].
55. Zeichner, S. L., J. Y. H. Kim, and J. C. Alwine. 1991. Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat. J. Virol. 65:2436-2444[Abstract/Free Full Text].
56. Zufferey, R., D. Nagy, R. J. Mandel, L. Naldini, and D. Trono. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15:871-875[Medline].

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  • Uesaka, T., Jain, S., Yonemura, S., Uchiyama, Y., Milbrandt, J., Enomoto, H. (2007). Conditional ablation of GFR{alpha}1 in postmigratory enteric neurons triggers unconventional neuronal death in the colon and causes a Hirschsprung's disease phenotype. Development 134: 2171-2181 [Abstract] [Full Text]  
  • Cui, C.-H., Uyama, T., Miyado, K., Terai, M., Kyo, S., Kiyono, T., Umezawa, A. (2007). Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation. Mol. Biol. Cell 18: 1586-1594 [Abstract] [Full Text]  
  • Greenberg, K. P., Geller, S. F., Schaffer, D. V., Flannery, J. G. (2007). Targeted Transgene Expression in Muller Glia of Normal and Diseased Retinas Using Lentiviral Vectors. IOVS 48: 1844-1852 [Abstract] [Full Text]  
  • Zhou, B. Y., Ye, Z., Chen, G., Gao, Z. P., Zhang, Y. A., Cheng, L. (2007). Inducible and Reversible Transgene Expression in Human Stem Cells After Efficient and Stable Gene Transfer. Stem Cells 25: 779-789 [Abstract] [Full Text]  
  • Masuda, H., Maruyama, T., Hiratsu, E., Yamane, J., Iwanami, A., Nagashima, T., Ono, M., Miyoshi, H., Okano, H. J., Ito, M., Tamaoki, N., Nomura, T., Okano, H., Matsuzaki, Y., Yoshimura, Y. (2007). Noninvasive and real-time assessment of reconstructed functional human endometrium in NOD/SCID/{gamma}Formula immunodeficient mice. Proc. Natl. Acad. Sci. USA 104: 1925-1930 [Abstract] [Full Text]  
  • Whitcomb, J. M., Huang, W., Fransen, S., Limoli, K., Toma, J., Wrin, T., Chappey, C., Kiss, L. D. B., Paxinos, E. E., Petropoulos, C. J. (2007). Development and Characterization of a Novel Single-Cycle Recombinant-Virus Assay To Determine Human Immunodeficiency Virus Type 1 Coreceptor Tropism. Antimicrob. Agents Chemother. 51: 566-575 [Abstract] [Full Text]  
  • Gray, P. C., Shani, G., Aung, K., Kelber, J., Vale, W. (2006). Cripto Binds Transforming Growth Factor {beta} (TGF-{beta}) and Inhibits TGF-{beta} Signaling. Mol. Cell. Biol. 26: 9268-9278 [Abstract] [Full Text]  
  • Vogel, M. J., Guelen, L., de Wit, E., Hupkes, D. P., Loden, M., Talhout, W., Feenstra, M., Abbas, B., Classen, A.-K., van Steensel, B. (2006). Human heterochromatin proteins form large domains containing KRAB-ZNF genes. Genome Res 16: 1493-1504 [Abstract] [Full Text]  
  • Irie, A., Harada, K., Tsukamoto, H., Kim, J.-R., Araki, N., Nishimura, Y. (2006). Protein kinase D2 contributes to either IL-2 promoter regulation or induction of cell death upon TCR stimulation depending on its activity in Jurkat cells. Int Immunol 18: 1737-1747 [Abstract] [Full Text]  
  • Philippe, S., Sarkis, C., Barkats, M., Mammeri, H., Ladroue, C., Petit, C., Mallet, J., Serguera, C. (2006). Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo. Proc. Natl. Acad. Sci. USA 103: 17684-17689 [Abstract] [Full Text]  
  • Yamamoto, T., Miyoshi, H., Yamamoto, N., Yamamoto, N., Inoue, J.-i., Tsunetsugu-Yokota, Y. (2006). Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages. Blood 108: 3305-3312 [Abstract] [Full Text]  
  • Jonsson, S. R., Hache, G., Stenglein, M. D., Fahrenkrug, S. C., Andresdottir, V., Harris, R. S. (2006). Evolutionarily conserved and non-conserved retrovirus restriction activities of artiodactyl APOBEC3F proteins. Nucleic Acids Res 34: 5683-5694 [Abstract] [Full Text]  
  • Mortellaro, A., Hernandez, R. J., Guerrini, M. M., Carlucci, F., Tabucchi, A., Ponzoni, M., Sanvito, F., Doglioni, C., Serio, C. D., Biasco, L., Follenzi, A., Naldini, L., Bordignon, C., Roncarolo, M. G., Aiuti, A. (2006). Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects. Blood 108: 2979-2988 [Abstract] [Full Text]  
  • Monse, H., Laufs, S., Kuate, S., Zeller, W. J., Fruehauf, S., Uberla, K. (2006). Viral determinants of integration site preferences of simian immunodeficiency virus-based vectors.. J. Virol. 80: 8145-8150 [Abstract] [Full Text]  
  • Hamamoto, S., Nishitsuji, H., Amagasa, T., Kannagi, M., Masuda, T. (2006). Identification of a Novel Human Immunodeficiency Virus Type 1 Integrase Interactor, Gemin2, That Facilitates Efficient Viral cDNA Synthesis In Vivo.. J. Virol. 80: 5670-5677 [Abstract] [Full Text]  
  • Ando, K., Yahata, T., Sato, T., Miyatake, H., Matsuzawa, H., Oki, M., Miyoshi, H., Tsuji, T., Kato, S., Hotta, T. (2006). Direct evidence for ex vivo expansion of human hematopoietic stem cells. Blood 107: 3371-3377 [Abstract] [Full Text]  
  • Yu, J. H., Schaffer, D. V. (2006). Selection of novel vesicular stomatitis virus glycoprotein variants from a Peptide insertion library for enhanced purification of retroviral and lentiviral vectors.. J. Virol. 80: 3285-3292 [Abstract] [Full Text]  
  • Wu, J. C., Spin, J. M., Cao, F., Lin, S., Xie, X., Gheysens, O., Chen, I. Y., Sheikh, A. Y., Robbins, R. C., Tsalenko, A., Gambhir, S. S., Quertermous, T. (2006). Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation. Physiol. Genomics 25: 29-38 [Abstract] [Full Text]  
  • Muotri, A. R., Nakashima, K., Toni, N., Sandler, V. M., Gage, F. H. (2005). Development of functional human embryonic stem cell-derived neurons in mouse brain. Proc. Natl. Acad. Sci. USA 102: 18644-18648 [Abstract] [Full Text]  
  • Sugiyama, S.-i., Shimada, N., Miyoshi, H., Yamauchi, K. (2005). Detection of Thyroid System-Disrupting Chemicals Using in Vitro and in Vivo Screening Assays in Xenopus laevis. Toxicol Sci 88: 367-374 [Abstract] [Full Text]  
  • Scott, B. B., Lois, C. (2005). Generation of tissue-specific transgenic birds with lentiviral vectors. Proc. Natl. Acad. Sci. USA 102: 16443-16447 [Abstract] [Full Text]  
  • Li, Q., Lu, Q., Estepa, G., Verma, I. M. (2005). Identification of 14-3-3{sigma} mutation causing cutaneous abnormality in repeated-epilation mutant mouse. Proc. Natl. Acad. Sci. USA 102: 15977-15982 [Abstract] [Full Text]  
  • Li, Q., Lu, Q., Bottero, V., Estepa, G., Morrison, L., Mercurio, F., Verma, I. M. (2005). Enhanced NF-{kappa}B activation and cellular function in macrophages lacking I{kappa}B kinase 1 (IKK1). Proc. Natl. Acad. Sci. USA 102: 12425-12430 [Abstract] [Full Text]  
  • Ikeda, H., Osakada, F., Watanabe, K., Mizuseki, K., Haraguchi, T., Miyoshi, H., Kamiya, D., Honda, Y., Sasai, N., Yoshimura, N., Takahashi, M., Sasai, Y. (2005). Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells. Proc. Natl. Acad. Sci. USA 102: 11331-11336 [Abstract] [Full Text]  
  • Nakayama, E. E., Miyoshi, H., Nagai, Y., Shioda, T. (2005). A Specific Region of 37 Amino Acid Residues in the SPRY (B30.2) Domain of African Green Monkey TRIM5{alpha} Determines Species-Specific Restriction of Simian Immunodeficiency Virus SIVmac Infection. J. Virol. 79: 8870-8877 [Abstract] [Full Text]  
  • Lucke, S., Grunwald, T., Uberla, K. (2005). Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors. J. Virol. 79: 9359-9362 [Abstract] [Full Text]  
  • Hanawa, H., Persons, D. A., Nienhuis, A. W. (2005). Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors. J. Virol. 79: 8410-8421 [Abstract] [Full Text]  
  • Hache, G., Liddament, M. T., Harris, R. S. (2005). The Retroviral Hypermutation Specificity of APOBEC3F and APOBEC3G Is Governed by the C-terminal DNA Cytosine Deaminase Domain. J. Biol. Chem. 280: 10920-10924 [Abstract] [Full Text]  
  • Kojima, H., Sasaki, T., Ishitani, T., Iemura, S.-i., Zhao, H., Kaneko, S., Kunimoto, H., Natsume, T., Matsumoto, K., Nakajima, K. (2005). STAT3 regulates Nemo-like kinase by mediating its interaction with IL-6-stimulated TGF{beta}-activated kinase 1 for STAT3 Ser-727 phosphorylation. Proc. Natl. Acad. Sci. USA 102: 4524-4529 [Abstract] [Full Text]  
  • Hirotani, T., Lee, P. Y., Kuwata, H., Yamamoto, M., Matsumoto, M., Kawase, I., Akira, S., Takeda, K. (2005). The Nuclear I{kappa}B Protein I{kappa}BNS Selectively Inhibits Lipopolysaccharide-Induced IL-6 Production in Macrophages of the Colonic Lamina Propria. J. Immunol. 174: 3650-3657 [Abstract] [Full Text]  
  • Motoyama, M., Yamazaki, S., Eto-Kimura, A., Takeshige, K., Muta, T. (2005). Positive and Negative Regulation of Nuclear Factor-{kappa}B-mediated Transcription by I{kappa}B-{zeta}, an Inducible Nuclear Protein. J. Biol. Chem. 280: 7444-7451 [Abstract] [Full Text]  
  • Rivera, S., Liu, L., Nemeth, E., Gabayan, V., Sorensen, O. E., Ganz, T. (2005). Hepcidin excess induces the sequestration of iron and exacerbates tumor-associated anemia. Blood 105: 1797-1802 [Abstract] [Full Text]  
  • Dodart, J.-C., Marr, R. A., Koistinaho, M., Gregersen, B. M., Malkani, S., Verma, I. M., Paul, S. M. (2005). Gene delivery of human apolipoprotein E alters brain A{beta} burden in a mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. USA 102: 1211-1216 [Abstract] [Full Text]  
  • Malik, P., Arumugam, P. I. (2005). Gene Therapy for {beta}-Thalassemia. ASH Education Book 2005: 45-50 [Abstract] [Full Text]  
  • Dittgen, T., Nimmerjahn, A., Komai, S., Licznerski, P., Waters, J., Margrie, T. W., Helmchen, F., Denk, W., Brecht, M., Osten, P. (2004). Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl. Acad. Sci. USA 101: 18206-18211 [Abstract] [Full Text]  
  • Coles, B. L. K., Angenieux, B., Inoue, T., Del Rio-Tsonis, K., Spence, J. R., McInnes, R. R., Arsenijevic, Y., van der Kooy, D. (2004). Facile isolation and the characterization of human retinal stem cells. Proc. Natl. Acad. Sci. USA 101: 15772-15777 [Abstract] [Full Text]  
  • Yamashita, D., Yamaguchi, T., Shimizu, M., Nakata, N., Hirose, F., Osumi, T. (2004). The transactivating function of peroxisome proliferator-activated receptor {gamma} is negatively regulated by SUMO conjugation in the amino-terminal domain. GENES CELLS 9: 1017-1029 [Abstract] [Full Text]  
  • Logan, A. C., Haas, D. L., Kafri, T., Kohn, D. B. (2004). Integrated Self-Inactivating Lentiviral Vectors Produce Full-Length Genomic Transcripts Competent for Encapsidation and Integration. J. Virol. 78: 8421-8436 [Abstract] [Full Text]  
  • Hofmann, A., Zakhartchenko, V., Weppert, M., Sebald, H., Wenigerkind, H., Brem, G., Wolf, E., Pfeifer, A. (2004). Generation of Transgenic Cattle by Lentiviral Gene Transfer into Oocytes. Biol. Reprod. 71: 405-409 [Abstract] [Full Text]  
  • Mitta, B., Weber, C. C., Rimann, M., Fussenegger, M. (2004). Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression. Nucleic Acids Res 32: e106-e106 [Abstract] [Full Text]  
  • Dai, C., McAninch, R. E., Sutton, R. E. (2004). Identification of Synthetic Endothelial Cell-Specific Promoters by Use of a High-Throughput Screen. J. Virol. 78: 6209-6221 [Abstract] [Full Text]  
  • Miyagi, S., Saito, T., Mizutani, K.-i., Masuyama, N., Gotoh, Y., Iwama, A., Nakauchi, H., Masui, S., Niwa, H., Nishimoto, M., Muramatsu, M., Okuda, A. (2004). The Sox-2 Regulatory Regions Display Their Activities in Two Distinct Types of Multipotent Stem Cells. Mol. Cell. Biol. 24: 4207-4220 [Abstract] [Full Text]  
  • Breckpot, K., Heirman, C., De Greef, C., van der Bruggen, P., Thielemans, K. (2004). Identification of New Antigenic Peptide Presented by HLA-Cw7 and Encoded by Several MAGE Genes Using Dendritic Cells Transduced with Lentiviruses. J. Immunol. 172: 2232-2237 [Abstract] [Full Text]  
  • Barzon, L., Boscaro, M., Palu, G. (2004). Endocrine Aspects of Cancer Gene Therapy. Endocr. Rev. 25: 1-44 [Abstract] [Full Text]  
  • Shibuya, K., Shirakawa, J., Kameyama, T., Honda, S.-i., Tahara-Hanaoka, S., Miyamoto, A., Onodera, M., Sumida, T., Nakauchi, H., Miyoshi, H., Shibuya, A. (2003). CD226 (DNAM-1) Is Involved in Lymphocyte Function-associated Antigen 1 Costimulatory Signal for Naive T Cell Differentiation and Proliferation. JEM 198: 1829-1839 [Abstract] [Full Text]  
  • Kuwata, H., Watanabe, Y., Miyoshi, H., Yamamoto, M., Kaisho, T., Takeda, K., Akira, S. (2003). IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF-{alpha} production in macrophages. Blood 102: 4123-4129 [Abstract] [Full Text]  
  • Tschan, M. P., Fischer, K. M., Fung, V. S., Pirnia, F., Borner, M. M., Fey, M. F., Tobler, A., Torbett, B. E. (2003). Alternative Splicing of the Human Cyclin D-binding Myb-like Protein (hDMP1) Yields a Truncated Protein Isoform That Alters Macrophage Differentiation Patterns. J. Biol. Chem. 278: 42750-42760 [Abstract] [Full Text]  
  • Haas, D. L., Lutzko, C., Logan, A. C., Cho, G. J., Skelton, D., Jin Yu, X., Pepper, K. A., Kohn, D. B. (2003). The Moloney Murine Leukemia Virus Repressor Binding Site Represses Expression in Murine and Human Hematopoietic Stem Cells. J. Virol. 77: 9439-9450 [Abstract] [Full Text]  
  • Lutzko, C., Senadheera, D., Skelton, D., Petersen, D., Kohn, D. B. (2003). Lentivirus Vectors Incorporating the Immunoglobulin Heavy Chain Enhancer and Matrix Attachment Regions Provide Position-Independent Expression in B Lymphocytes. J. Virol. 77: 7341-7351 [Abstract] [Full Text]  
  • Dirac, A. M. G., Bernards, R. (2003). Reversal of Senescence in Mouse Fibroblasts through Lentiviral Suppression of p53. J. Biol. Chem. 278: 11731-11734 [Abstract] [Full Text]  
  • Marr, R. A., Rockenstein, E., Mukherjee, A., Kindy, M. S., Hersh, L. B., Gage, F. H., Verma, I. M., Masliah, E. (2003). Neprilysin Gene Transfer Reduces Human Amyloid Pathology in Transgenic Mice. J. Neurosci. 23: 1992-1996 [Abstract] [Full Text]  
  • Kubo, S., Mitani, K. (2003). A New Hybrid System Capable of Efficient Lentiviral Vector Production and Stable Gene Transfer Mediated by a Single Helper-Dependent Adenoviral Vector. J. Virol. 77: 2964-2971 [Abstract] [Full Text]  
  • Roszkowski, J. J., Yu, D. C., Rubinstein, M. P., McKee, M. D., Cole, D. J., Nishimura, M. I. (2003). CD8-Independent Tumor Cell Recognition Is a Property of the T Cell Receptor and Not the T Cell. J. Immunol. 170: 2582-2589 [Abstract] [Full Text]  
  • Xia, Y., Pao, G. M., Chen, H.-W., Verma, I. M., Hunter, T. (2003). Enhancement of BRCA1 E3 Ubiquitin Ligase Activity through Direct Interaction with the BARD1 Protein. J. Biol. Chem. 278: 5255-5263 [Abstract] [Full Text]  
  • Coleman, J. E., Huentelman, M. J., Kasparov, S., Metcalfe, B. L., Paton, J. F. R., Katovich, M. J., Semple-Rowland, S. L., Raizada, M. K. (2003). Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo. Physiol. Genomics 12: 221-228 [Abstract] [Full Text]  
  • Gough, P. J., Raines, E. W. (2003). Gene therapy of apolipoprotein E-deficient mice using a novel macrophage-specific retroviral vector. Blood 101: 485-491 [Abstract] [Full Text]  
  • Mitta, B., Rimann, M., Ehrengruber, M. U., Ehrbar, M., Djonov, V., Kelm, J., Fussenegger, M. (2002). Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction. Nucleic Acids Res 30: e113-e113 [Abstract] [Full Text]  
  • Lai, Z., Han, I., Park, M., Brady, R. O. (2002). Design of an HIV-1 lentiviral-based gene-trap vector to detect developmentally regulated genes in mammalian cells. Proc. Natl. Acad. Sci. USA 99: 3651-3656 [Abstract] [Full Text]  
  • Pfeifer, A., Ikawa, M., Dayn, Y., Verma, I. M. (2002). Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc. Natl. Acad. Sci. USA 99: 2140-2145 [Abstract] [Full Text]  
  • Konetschny, C., Holzer, G. W., Falkner, F. G. (2002). Retroviral Vectors Produced in the Cytoplasmic Vaccinia Virus System Transduce Intron-Containing Genes. J. Virol. 76: 1236-1243 [Abstract] [Full Text]  
  • Cui, Y., Golob, J., Kelleher, E., Ye, Z., Pardoll, D., Cheng, L. (2002). Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells. Blood 99: 399-408 [Abstract] [Full Text]  
  • Borok, Z., Harboe-Schmidt, J. E., Brody, S. L., You, Y., Zhou, B., Li, X., Cannon, P. M., Kim, K.-J., Crandall, E. D., Kasahara, N. (2001). Vesicular Stomatitis Virus G-Pseudotyped Lentivirus Vectors Mediate Efficient Apical Transduction of Polarized Quiescent Primary Alveolar Epithelial Cells. J. Virol. 75: 11747-11754 [Abstract] [Full Text]  
  • Pang, S., Kang, M. K., Kung, S., Yu, D., Lee, A., Poon, B., Chen, I. S. Y., Lindemann, B., Park, N.-H. (2001). Anticancer Effect of a Lentiviral Vector Capable of Expressing HIV-1 Vpr. Clin. Cancer Res. 7: 3567-3573 [Abstract] [Full Text]  
  • Moreau-Gaudry, F., Xia, P., Jiang, G., Perelman, N. P., Bauer, G., Ellis, J., Surinya, K. H., Mavilio, F., Shen, C.-K., Malik, P. (2001). High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors. Blood 98: 2664-2672 [Abstract] [Full Text]  
  • Lewis, B. C., Chinnasamy, N., Morgan, R. A., Varmus, H. E. (2001). Development of an Avian Leukosis-Sarcoma Virus Subgroup A Pseudotyped Lentiviral Vector. J. Virol. 75: 9339-9344 [Abstract] [Full Text]  
  • Wolfgang, M. J., Eisele, S. G., Browne, M. A., Schotzko, M. L., Garthwaite, M. A., Durning, M., Ramezani, A., Hawley, R. G., Thomson, J. A., Golos, T. G. (2001). Rhesus monkey placental transgene expression after lentiviral gene transfer into preimplantation embryos. Proc. Natl. Acad. Sci. USA 98: 10728-10732 [Abstract] [Full Text]  
  • Pfeifer, A., Brandon, E. P., Kootstra, N., Gage, F. H., Verma, I. M. (2001). Delivery of the Cre recombinase by a self-deleting lentiviral vector: Efficient gene targeting in vivo. Proc. Natl. Acad. Sci. USA 10.1073/pnas.201415498v1 [Abstract] [Full Text]  
  • Kowolik, C. M., Hu, J., Yee, J.-K. (2001). Locus Control Region of the Human CD2 Gene in a Lentivirus Vector Confers Position-Independent Transgene Expression. J. Virol. 75: 4641-4648 [Abstract] [Full Text]  
  • Berkowitz, R., Ilves, H., Lin, W. Y., Eckert, K., Coward, A., Tamaki, S., Veres, G., Plavec, I. (2001). Construction and Molecular Analysis of Gene Transfer Systems Derived from Bovine Immunodeficiency Virus. J. Virol. 75: 3371-3382 [Abstract] [Full Text]  

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