Human cytomegalovirus plasmid-based amplicon vector system for gene therapy
© Mahmood et al; licensee BioMed Central Ltd. 2005
Received: 28 August 2004
Accepted: 26 January 2005
Published: 26 January 2005
We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.
HCMV is a member of the betaherpesvirus family [42, 48]. Other members of this family include human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), and all are widely distributed in human populations. During productive replication, the 230 kilobase pair (kbp) viral genome replicates by a rolling circle mechanism, which generates long head-to-tail concatemers that are cleaved to unit length and packaged in capsids. The state of the HCMV genome during latency remains unidentified and is likely to be circular and extrachromosomal . The HCMV genome has been detected in cells within the hematopoietic lineage as early as CD34+ progenitors and up through differentiated macrophages [23, 29, 38, 54].
Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level [13, 18, 31, 43, 52, 67]. Naturally occurring defective HSV viruses and laboratory derived HSV amplicon vectors are composed of head-to-tail tandem reiterations of subgenomic regions containing a functional origin of DNA replication (OriSor OriL) and a DNA cleavage/packaging signal [3, 4, 30, 57, 60–62]. These two cis-acting functions can be relatively small ranging from ca. 90–150 base pairs (bp) for the ori and ca. 250–300 bp for the a sequence. The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp [1, 2, 24, 37]. HCMV is unique among the herpesviruses in not having an origin binding protein homolog that is required for DNA replication . The HCMV a sequence varies in size from ca. 550 bp to 762 bp, however, the conserved pac-1 and pac-2 cis-elements which determine the sites for cleavage of replicated viral DNA are present [15, 28, 58, 64, 65].
In contrast to HSV, HCMV does not efficiently produce defective virus genomes, this difference may be related to the distinct biology of the two viruses . However two reports described the identification of what may potentially be HCMV defective viruses created by serial high multiplicity passage [47, 59]. These reports characterized HCMV defectives as very large subgenomic DNA molecules, in some cases extending over two thirds of the genome. In addition to these replication defective HCMV viruses, a recent report by Borst et al. 2003 , described the construction of an HCMV amplicon. In this report we further utilized the HCMV amplicon for gene delivery to human CD34+ cells.
HCMV infects cells of the hematopoietic lineage [34, 38, 39, 55, 68]. Viral genomes can be found in CD34+ cells from seropositive individuals and granulocyte-macrophage progenitors and differentiated macrophages can be infected experimentally . We were interested in determining whether the tropism of HCMV can be exploited to construct defective HCMV virus vectors (amplicons) targeted to hematopoietic stem cells. The general feasibility of such an approach for other cell types has been shown using other herpesviruses, e.g. HSV, EBV, and HHV-7 [20, 25, 26, 30, 35, 49, 70, 71].
Cells and virus
HCMV Toledo (passage 8, from Dr. S. Plotkin, Aventis Pasteur, Doylestown, PA), and HCMV TownevarRIT (passage 134, from Dr. Plotkin via Dr. Ed Mocarski, Stanford University), were propagated in human fibroblasts (HF) cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, Kans.). Recombinant HCMV, RC2.7EGFP, expressing enhanced green fluorescent protein (EGFP) (Clonetech, Palo Alto, CA), under the control of the major early 2.7 promoter, was constructed by cotransfection of plasmid pEAG2.7EGFP with a set of overlapping cosmid clones derived from HCMV Toledo (G.M. Duke, unpublished data).
Generation of viral stocks containing amplicons
Plasmid DNA was transfected by CaPO4 precipitation of approximately 4 μg of Tn9-8 amplicon DNA. The Tn9-8 DNA was transfected into approximately 1 × 106 passage 16 human fibroblast (HF) cells. At 24 hours post transfection, the cells were infected with CMV Towne at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Fresh medium was added to cells four days after infection and cells were harvested at 6 to 7 days post infection as described previously (Spaete and Frenkel, 1982). Virus stocks are prepared by three freeze-thaw cycles. Serial passages of amplicon-containing viral stocks on fresh HF cells were superinfected with CMV Towne as a helper virus at a MOI of 1.
Southern blot analysis
Viral DNAs were digested with restriction enzyme, electrophoresed in 0.8% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Corp.), (Maniatis et al., 1989), and immobilized with a UV Crosslinker 1000 (Hoefer Scientific Instruments, San Francisco, CA). DNA on the membrane was probed with fluorescein-labeled pUC9 DNA using conditions previously described (Spaete and Mocarski, 1985).
Isolation of CD34+cells and infection with CMV
The isolation of cord blood CD34+ stem cells was carried out by All Cells Inc. (Berkeley, CA) using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA). The positive selection of the CD34+ cells was carried out using hapten-conjugated antibody to CD34+ followed by anti-hapten antibody coupled to MACS Microbeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field. The purity of the CD34+ population was >95% as analyzed by flow cytometry. The purified CD34+ cells were suspended in Iscove's modified Dulbecco's Minimal Essential Medium containing 5% fetal bovine serum. 2 × 105 CD34+ cells were used for each infection with TN9-8GF5 amplicon containing stocks, RC2.7EGFP virus, CMV Towne virus, or uninfected cell control. The cells mixed with virus were centrifuged at 500 × g for 10 mins at room temperature and were then placed in 37°C water bath for one hour. Following this the cells were cultured in 6-well cell culture plates (Costar) for 18–72 hours. At the end of the incubation the cells were harvested for CD34 staining.
EGFP expression and immunostaining for flow cytometric analysis
Amplicon containing viral stocks prepared from passage 1 were used to infect HF or human CD34+ cells maintained in 12 well culture plates. At 24 hour intervals post infection, the wells were observed for EGFP expression with a Nikon TE2000 microscope under UV illumination. Immunostaining for CD34+ cells was done using Phycoerythrin (PE)-conjugated anti-CD34 antibody (Becton Dickinson, San Jose, CA). Infected or control cells were incubated with 20 μl of PE-labeled anti-CD34 antibody for 45 minutes at room temperature and subsequently were washed twice with PBS containing 0.1% BSA. The cells were directly analyzed for EGFP and CD34+ staining on a FACSCalibur instrument (Becton Dickinson, San Jose, CA), at 18 and 36 hours post infection.
In order to exploit the natural tropism of HCMV for cells of the hematopoietic lineage, in a nonlytic manner, an HCMV amplicon i.e. a plasmid containing the HCMV oriLyt and a sequences was constructed. Theoretically, due to the large size of the HCMV genome, an amplicon derived from this virus should be able to carry the large DNA inserts and be capable of efficient introduction into hematopoietic cells by infection.
Heterogeneity at oriLyt
During analysis of cosmid clones of HCMV strain Towne, sequence heterogeneity was observed in the EcoRI E fragment of Towne that was not present in the Toledo strain . The EcoRI E region spans in part the complex oriLyt region [1, 2, 24, 37]. Sequences in a 1.2 kbp repeat fragment were shown to give rise to the heterogeneity observed at this locus in the Towne genome (Figure 1). The coordinates of a single repeat unit starts at nucleotide 94,561 relative to the AD169 sequence and end at nucleotide 95,807 . This segment can repeat at least three times in Towne strains from different passage histories (Fig. 1). This heterogeneity is different from the 189 bp repeat region previously described for the Towne strain oriLyt which occurs near the BamHI sites in Figure 1 (nt 93337–93525 relative to AD169), [11, 12]. Since Towne replicates to relatively high titers in cell culture, it was deemed advantageous to incorporate this heterogeneity in the origin containing sequences to be used in the amplicon construct.
Construction of the HCMV amplicon
Generation of viral stocks containing amplicons
Packaged defective viral genomes derived from Tn9-8 or a derivative containing a selectable marker (Tn9-8-gpt), were serially passaged in HF cells. Defective viruses could be detected at passage 3 when probed with plasmid pUC9; however, the copy number appeared to diminish upon serial passage (not shown). Selection with mycophenolic acid on Tn9-8-gpt amplicons did not enhance recovery.
Rescue of monomeric repeat units in bacteria
Expression of heterologous genes in an HCMV amplicon
To test the utility of the HCMV amplicon in gene therapy or gene delivery, we used packaged amplicons in viral stocks to infect and deliver an expressed gene into human CD34+ progenitor cells. Viral stocks containing amplicons carrying EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter prepared from passage 0 and passage 1 were used to infect CD34+ cells derived from cord blood. Starting at 24 h after infection, CD34+ cells were examined for EGFP expression by fluorescent microscopy. EGFP expression was observed in TN9-8GF5 amplicon-infected CD34+ cells starting at 24 h post-infection. The cells remained positive for EGFP expression for more than 96 hrs, at which point the cells were terminated (Figure 6).
We have shown that a replication-defective virus vector system that is derived from HCMV is capable of delivering and expressing foreign genes in infected primary cells including progenitor stem cells such as human CD34+ cells. Further improvement and optimization of the system offers the potential to deliver gene-based therapies to multipotent cells.
Advantages for use of the HCMV amplicon
Foremost among the advantages of the vector system we have described is the potential ability to efficiently infect and deliver genetic information to hematopoietic stem cells (CD34+) and other dividing and non-dividing cell types which may support HCMV infection [34, 38, 39, 55, 68]. Genetic hematological disorders such as thalassemias and sickle-cell anemia and other hemaglobinopathies could therefore be targeted for therapy with this strategy. Another potential advantage for the system is that vector DNA could possibly be maintained as an episome with minimal concern for the potential consequences of random integration of vector DNA (i.e. activation of oncogenes or inactivation of tumor suppressor genes). In order to insure efficient segregation as an episome, the EBV latent replication origin, oriP, and the transactivator, EBNA-1, could be added as was previously shown for another hybrid herpesvirus vector . However such a modification may not be necessary because HCMV genomes appear to be carried continuously in cells of hematopoietic origin in infected individuals. Yet another potential advantage as with other herpesviral vectors, is that the HCMV vector system should have the capacity for very large inserts.
Infection of CD34+ cells with HCMV
The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both in vivo and in vitro . Furthermore, hematopoietic stem cells are also reported as a site for HCMV latency. Efficient transduction of human CD34+ cells with retroviral and non-viral vectors has been unsatisfactory due to the lack of maintenance of high levels of expression of the transgene following engraftment of the engineered cells . The HCMV MIE promoter may not be the right promoter for optimal expression in a CD34+ cells, since it has been shown that in the context of a lentiviral-based gene transfer system this promoter appeared to function less efficiently due to a cell-type specific expression defect . The approaches to improving the efficiency of gene transfer into human cells have focused on improving gene delivery vectors and optimizing ex vivo culture conditions, which preserve the developmental properties of the stem cells [14, 22]. Umbilical cord blood is recognized as a rich source of hematopoietic CD34+ stem cells . In our experiments we used cord blood derived CD34+ cells for infection with HCMV amplicon containing stocks or HCMV-EGFP virus. However, bone marrow derived CD34+ cells have also been shown to be infectable in vitro with HCMV . Gentry & Smith , reported a progressive loss of primitive cell properties including a reduction of CD34 expression upon in vitro culture of cord blood derived CD34+ cells. In a separate study, cord blood derived CD34+ cells cultured with IL-3 in vitro showed a progressive decline of the CD34+ population and more differentiated cells originating in the CD34(-) population . In our experiments with >95% pure cord blood derived CD34+ cell population, a loss of CD34 expression in a small percent population (9–12%) of stem cells upon in vitro culture has been observed. EGFP expression was also seen in the CD34(-) population (Fig 7a, 7b, &7c). It is possible that HCMV infection of CD34 cells could induce cell differentiation and loss of primitive properties including reduction of CD34 expression.
Further studies of HCMV infection in CD34 cells will help in defining whether CD34+ infected cells undergo cell differentiation by increased expression of other markers such as CD33, CD38, HLA-DR or cytokines. It is also relevant to note here that HCMV virus carries homolog sequences for HLA-related and cytokine-related molecules and infection can induce cellular cytokines [5, 8, 19, 32, 44, 46, 50, 66]. The HCMV amplicons contain only the cis-acting ori and packaging sequences, and have no structural gene sequences. However, amplicon containing viral stocks are a mixture with HCMV replication competent helper virus. HCMV induced cell-differentiating effect, if any, might be minimized using a helper virus-free amplicon system. In this regard, it should be possible to test a number of strategies to prepare helper virus-free stocks [17, 63]. These preparations would be useful for therapeutic applications in immuno-compromised patients.
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