Adenoviral vectors for transient gene expression in human primitive hematopoietic cells: Applications and prospects

Experimental Hematology 35 (2007) 343–349

Adenoviral vectors for transient gene expression in human primitive hematopoietic cells: Applications and prospects Marcus Ja¨ra˚sa, Ann C.M. Bruna, Stefan Karlssona, and Xiaolong Fana,b a

Section of Molecular Medicine and Gene Therapy, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund, Sweden; bSection of Immunology, Lund University, Lund, Sweden (Received 10 October 2006; revised 10 October 2006; accepted 7 November 2006)

The proliferation and differentiation of primitive hematopoietic cells is tightly controlled by a number of signaling pathways. Transient blockage or enhancement of these signaling pathways may provide a new approach to manipulate the proliferation and differentiation of primitive hematopoietic cells. Adenoviral vectors have in recent years emerged as powerful tools for transient gene expression in human primitive hematopoietic cells. Important advantageous properties of adenoviral vectors include: feasible production of high-titer vector preparations, high efficiency in transducing both quiescent and actively dividing cells, high levels of transient gene expression, and a lack of mutagenic properties associated with integrating vectors. Progress in adenoviral fiber retargeting was recently demonstrated to enable high gene transfer efficiency into nondividing human CD34+ cells and nonobese diabetic/severe combined immunodeficient mouse bone marrow repopulating cells (SRCs), via the ubiquitously expressed CD46 as a cellular receptor. Importantly, fiber-retargeted adenoviral vectors can be engineered to report gene expression in single living CD34+ cells, thereby facilitating the isolation and characterization of SRCs and its downstream progenitors based on intrinsic signaling pathways. This review focuses on the current progress and the potential future applications of adenoviral gene transfer into human primitive hematopoietic cells and leukemic cells. Ó 2007 International Society for Experimental Hematology. Published by Elsevier Inc.

Viral vectors are widely used for transgene expression studies in different organ systems. For human primitive hematopoietic cells, oncoretroviral vector systems are most commonly used. However, recent clinical and experimental studies have shown that insertional mutagenesis following oncoretroviral integrations into hematopoietic stem cells (HSCs) might lead to clonal dominance followed by leukemic transformation [1–3]. Thus, for purposes when a transient gene expression is sufficient, nonintegrating viral vectors are preferred. Due to their episomal nature, the adenoviral vector genome normally does not integrate into the host cell genome and therefore does not cause insertional mutagenesis. Furthermore, adenoviral vectors can allow efficient gene transfer into both actively dividing and also quiescent cells, which is frequently followed by high levels of transient gene expression. In recent years, adenoviral vector fiber retargeting has been successfully performed, Offprint requests to: Xiaolong Fan, M.D., Ph.D., Section of Molecular Medicine and Gene Therapy, BMC A12, 221 84 Lund, Sweden; E-mail: [email protected]

allowing high gene transfer efficiency into human primitive hematopoietic cells [4–7]. Of particular importance, Ad5based vectors with fibers retargeted to Ad35 tropism (Ad5F35 vectors) have proven to transduce primitive progenitor cells and self-renewing nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) with high efficiency [7,8]. Thus, adenoviral vectors have emerged as powerful tools for studying and manipulating the function of human candidate HSCs.

Adenoviral vector developments open up new possibilities for efficient gene transfer into human primitive hematopoietic cells The most widely used adenoviral vectors are based on the Ad5 serotype, belonging to the adenovirus subgroup C. Ad5-based vectors have been considered to be rather inefficient as gene transfer tools for primitive hematopoietic cells [9], due to a paucity of the coxsackie and adenovirus receptor (CAR) on these cells, which is essential for Ad5 binding to host cells [10]. However, early studies showed

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that a fraction of primitive hematopoietic cells could be transduced at high multiplicity of infection (MOI) [11– 13], but this is associated with nonspecific cytotoxicity in transduced cells [12]. Several approaches to overcome CAR deficiency have been explored, including (see Fig. 1): 1) the use of bispecific conjugates with high affinity for both the fiber proteins and a cell surface receptor [14–17]; 2) genetic modification of the fiber by insertion of nonviral targeting sequences [18– 21]; 3) swapping the fiber knob domain to a knob domain from another serotype (i.e., fiber retargeting) [4,5,22,23]; 4) the use of polycationic reagents to allow nonreceptor mediated uptake of viral particles [24–26]; and 5) treatment of CD34þ cells with FR901228, a histone deacetylase inhibitor, for upregulation of CAR expression [27]. Importantly, optimization of viral transduction efficiency by polycationic reagents (4) resulted in proof-of-principle studies, demonstrating that SRCs could in fact be transduced by Ad5-based vectors [24]. The most successful approach so far has been the utility of fiber-retargeted vectors (see Fig. 2) (3) as these vectors, in contrast to those described in (2), can be produced without reduced titers and can efficiently transduce hematopoietic cells. For (1) and (4), relatively high MOIs were required, which is naturally associated with increased risks of nonspecific cytotoxicity in targeted cells. Histone deacetylase inhibitors (5) can most likely affect the expression of genes other than CAR, thereby potentially compromising the functionality of human primitive hematopoietic cells. Fiber-swapping to adenoviruses of subgroup B (e.g., Ad11, Ad35) seems to be the most efficient approach for

Figure 1. Different strategies for increasing the transduction efficiency. The use of bispecific conjugates (shown in orange) with high affinity for both the fiber proteins and a cell surface receptor (1). Genetic modification of the fiber by insertion of nonviral targeting sequences (shown in blue) (2). Swapping the fiber knob domain to a knob domain (shown in orange) from another serotype (3). The use of polycationic reagents (positively charged) to allow non-receptor-mediated uptake of viral particles (4). Treatment of CD34þ cells with FR901228, a histone deacetylase inhibitor, for upregulation of CAR expression.

Figure 2. Attachment of fiber-retargeted adenoviral vectors to CD34þ cells. By swapping the knob domain of the fiber gene to that of the subgroup B adenovirus species, high transduction efficiency of CD34þ cells can be achieved. The knob domain of the subgroup B adenovirus binds the CD46 receptor, which is highly expressed on human cells.

transduction of human hematopoietic cells with a primitive immunophenotype [4–6], whereby the ubiquitously expressed CD46 receptor is exploited for binding to target cells [28,29]. Importantly, we demonstrated that Ad35 tropism vectors can efficiently transduce primary and secondary SRCs with self-renewal capacity [7,8]. Recently, entirely new non-fiber-modified adenoviral vector systems based on the Ad35 serotype genome have been developed [30,31]. In addition to the CAR-independent gene delivery mechanisms, cells transduced by Ad35-based vectors are less likely to be eliminated by a preexisting anti-Ad5 immunity [31]. In addition, a recent evaluation of adenoviral vectors containing serotype 35 fibers for vaccination revealed that overall, compared with Ad5 vectors, fiberretargeted Ad5/35 vectors have a better safety profile, reflected by lower serum levels of proinflammatory cytokines following injections into CD46 transgenic mice [32]. Furthermore, a promising toxicology study in baboons demonstrated that Ad vectors possessing Ad35 or Ad11 fibers have a better safety profile after intravenous injection compared with conventional Ad5-based vectors [33]. Thus, Ad35 and Ad5/35 vectors seem to be superior for gene therapy approaches compared with conventional Ad5 vectors.

Production and design of adenoviral vector constructs for high-level transgene expression in human primitive hematopoietic progenitors Most adenoviral vectors developed for gene deliveries into human primitive hematopoietic cells are first-generation vectors with the expression cassette engineered into the E1 or E3 region. The recombinant, replication-deficient adenoviral vectors are produced in 293 cells, providing the E1A gene in trans, and purified by cesium chloride density gradient ultracentrifugation or ion-exchange chromatography. The AdEasy system is frequently used for generation of

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the plasmid encoding the recombinant adenoviral vector genome via homologous recombination between the shuttle plasmid and the adenoviral plasmid in the Escherichia coli strain BJ5183 (see Fig. 3). The vectors generated with the AdEasy system can accommodate an expression cassette between 7 and 8 kilobase pairs (kb) [34]. By a fiber modification to Ad35 fiber specificity, the cloning capacity can be extended to about 9 kb [23]. For sophisticated purposes such as erythroid-specific gene expression under the control of the 22-kb b-globin locus control region, gutless adenoviral vectors devoid of all viral genes are employed [35]. Studies from our laboratory and others have suggested that in the context of adenoviral vector-mediated gene delivery into human primitive hematopoietic progenitor cells, specific regulatory sequences are required for high levels of transgene expression. In CD34þ cells, the intron containing cytomegalovirus promoter/enhancer, the elongation factor 1a promoter, the CA promoter, the murine stem cell long terminal repeats promoter, and the murine pgk-1 promoter were demonstrated to allow high levels of transgene expression [36,37], whereas the LTR of the Rous sarcoma virus and the SR-a promoter have proven to be inefficient for gene expression in CD34þ cells [25]. However, because CD34þ cells are heterogeneous, phenotypic data on transgene expression in CD34þ cells need to be verified in functional assays for the confirmation of gene delivery into HSCs and their progenitors. So far, adenoviral vectormediated gene transfer into SRCs has been reported only when the murine pgk-1 promoter was used for controlling transgene expression [7,8,24]. The relative levels of transgene expression from the pgk-1 promoter-controlled ex-

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pression cassette are also dependent on the 30 regulatory sequences. By switching the SV40 polyadenylation signal to the b-globin gene intron 2 and polyadenylation signal, the relative level of transgene expression in CD34þ cells could be markedly increased [36]. This effect was specific to human primitive hematopoietic cells, as an opposite effect was observed in HeLa cells and K562 cells [36]. Because current adenoviral vector-mediated gene delivery protocols can achieve transgene expression in only about 50% of human primitive cells [7,37], successfully transduced cells need to be identified and isolated for studying the consequences of transgene expression. This is normally achieved by cloning of a reporter gene (e.g., GFP) together with the functional gene into the same vector, either using a bicistronic expression cassette [38] or two expression cassettes in the same vector [34,39]. However, both strategies are compromised by disadvantages. In adenoviral vectors with bicistronic expression cassettes, the reporter gene is routinely placed as the second gene following an internal ribosome entry site (IRES). As such, the reporter gene is frequently expressed at much lower levels compared with the first functional gene, thereby limiting the identification of successfully transduced cells. In adenoviral vectors encoding two expression cassettes, both cassettes are frequently driven by the same promoter;, however, such vector genomes are prone to DNA rearrangement during the plasmid expansion in the recombination competent E. coli BJ5183 strain or in the viral expansion steps in 293 cells. Thus, if the marker gene expression is sufficient from the bicistronic expression cassette, we suggest that this alternative should be the first choice.

Figure 3. Design of adenoviral vectors for optimal transient gene expression. The gene of interest (Gene X) is cloned into the pShuttle plasmid in between the promoter (yellow) and IRES sequence (gray), which is followed by a marker gene such as GFP (green). The pShuttle is recombined with the adenoviral genomic plasmid before viral vectors can be generated in 293 cells. The fiber gene sequence in the genomic plasmid is marked.

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Transduction of human primitive hematopoietic cells To preserve the primitive hematopoietic cells during the transduction protocol, it is important to establish a short transduction protocol with serum-free medium and minimal cytokine stimulation, as serum-containing media and multiple cytokine stimulation induce differentiation of primitive hematopoietic cells [40]. In our laboratory, we routinely infect overnight cultured CD34þ cells with fiber-retargeted Ad5F35 vectors at an MOI of 100. The use of higher MOIs is not recommended due to cytotoxicity in CD34þ cells at high vector concentrations. After 6 to 7 hours of incubation, the medium is replaced with fresh medium. During the culture and transduction period, either thrombopoietin (TPO) alone or a cytokine combination of TPO, stem cell factor, and Flt-3 ligand can be used to support the primitive hematopoietic cells [41,42]. If the vector contains a marker gene such as GFP, the percentage of GFPþ cells can be measured 48 hours posttransduction using flow cytometry and the GFPþ cells can be sorted for further studies. Sorted GFPþ cells can be functionally studied in various hematopoietic assays (e.g. cell proliferation, colony-forming, cell-cycle analysis, NOD/SCID mouse transplantation assays) and for changes in global gene expression (see Fig. 4).

Applications for adenoviral vector-mediated gene delivery into human primitive hematopoietic cells Adenoviral gene transfer into HSCs and progenitor cells has many potential applications: by overexpression of immune response-modulating molecules, it can be used for immunotherapies against leukemias; by overexpression of ‘‘stemness’’ genes, it can potentially be used to expand HSCs or manipulate their differentiation without permanently changing their genomes. Moreover, adenoviral reporters can be used to detect and elucidate crucial signaling pathways in living cells, which can facilitate the

isolation and functional assessment of human primitive hematopoietic cells. Transgene overexpression studies Primitive hematopoietic cells are hierarchically organized. The most primitive long-term reconstituting HSCs have extensive self-renewal capacity, although this property is gradually lost upon differentiation to short-term HSCs and lineage-restricted progenitor cells. As self-renewal and commitment to certain lineages depend on the activation and inactivation of different signaling pathways, it can be envisaged that adenoviral vector-mediated transient gene expression can be used to interfere with these signaling pathways and thereby to manipulate the proliferation and differentiation capacity of human primitive hematopoietic progenitor cells. Following adenoviral vector-mediated gene delivery into human CD34þ cells, the transgene expression peaks 2 to 4 days posttransduction and is lost around 2 weeks posttransduction depending on the proliferation rate of the cells [7,36]. By functional characterization of the sorted GFPexpressing cells following adenoviral vector infection, we and others have demonstrated that the Ad5 vector will preferentially infect actively dividing, relatively differentiated progenitors, whereas the Ad5F35 vectors can efficiently infect both dividing and nondividing progenitor cells, including self-renewing SRCs [7,8]. Because the adenoviral life cycle is episomal, it has been speculated that adenoviral vectors would be ideal for HSC expansion in vitro without permanently affecting the genomes of the HSCs and their progenies. By transient overexpression of the epidermal growth factor receptor in mobilized peripheral blood CD34þ cells, it was demonstrated that progenitors with colony-forming capacity could be expanded following stimulation with the epidermal growth factor [13]. Moreover, adenoviral vector-mediated overexpression of a dominant negatively acting mutant of the transforming growth factor beta (TGF-b) type II receptor was shown to block the

Figure 4. Adenoviral vector-mediated transient gene expression in human primitive hematopoietic cells. CD34þ cells can be efficiently infected by Ad5F35 vectors encoding functional genes and GFP at an MOI of 100. At 48 hours posttransduction, GFP-expressing cells can be sorted by flow cytometry and assessed in a variety of functional assays to reveal the functional properties of the transduced cells and changes in their gene expression.

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autocrine and paracrine TGF-b signaling in primitive human hematopoietic progenitor cells and enhance their survival and proliferation at the single cell level [38]. However, it is important to keep in mind that the transgene expression from adenoviral vectors is usually rather high initially and then declines over time. In addition, compared with retroviral vector-mediated gene delivery, fiberretargeted Ad5F35 vectors or the ordinary Ad5 vectors may transduce different subsets of CD34þ cells. Therefore, unexpected findings can be observed following adenoviral vector-mediated gene transfer. For example, high adenoviral HOXB4 overexpression led to myeloid differentiation of CD34þ cells [39], and relatively lower retroviral HOXB4 overexpression in CD34þ cells led to expansion of SRCs with no detectable alterations in lymphomyeloid reconstitution [43]. Gene expression reporting studies in single living primitive hematopoietic cells HSCs and their immediate progenitor cells have so far been prospectively isolated primarily by cell surface marker expression followed by assessments of their functional properties in various assays [44]. This strategy has been successful for murine HSCs and progenitor cells. However, the identification of their human counterparts has proven to be more challenging. We hypothesize that an alternative strategy to directly isolate HSCs and their downstream progenitors could be to use adenoviral vectors for reporting of intrinsic signaling pathways in single living primitive hematopoietic cells. Previous studies have shown that adenoviral vectors can be engineered for tissue- or celltype-specific transgene expression [45,46]. In our laboratories, we have developed Ad5F35 vectors driving destabilized GFP (dGFP) [47] under the control of the human telomerase reverse transcriptase (hTERT) promoter for reporting hTERT expression in cord blood CD34þ cells [8,48]. The telomerase enzyme complex consists of two essential components, the catalytic protein component hTERT and the telomerase reverse RNA component (TERC). Telomerase activity is suggested to be differentiation and proliferation status dependent in the human hematopoietic system [49,50]. In patients, mutations in hTERC have been linked to the rare autosomal dominant form of dyskeratosis congenita disorder and also in sporadic cases of aplastic anemia [51–56]. These patients usually manifest a bone marrow failure syndrome prior to age 50, suggesting an HSC defect as the primary cause behind the disease. Because mtert knockout mice show a premature exhaustion of HSCs upon serial transplantations, it was speculated that the level of mtert expression in HSCs might limit their long-term survival [57]. However, the life span of HSCs could not be extended by mtert overexpression in transgenic mice, demonstrating that mtert overexpression alone is not sufficient to prolong the life span of HSCs [58]. However, the implications from such studies in inter-

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preting telomerase function in human primitive hematopoietic cells are complicated by species differences, attributed to the relatively longer telomeres in inbred mouse strains compared with humans. In this hTERT-reporting vector [48], insulator sequences from the chicken b-like globin gene [59] were inserted in front of the hTERT promoter to shield it from potential effects of viral enhancer elements overlapping with the inverted terminal repeats and the packaging signal of the adenovirus [60]. As such, single living primitive hematopoietic cells with upregulated hTERT expression could be isolated. We demonstrated that hTERT expression is not detectable in self-renewing SRCs but is upregulated in a transition from long-term to short-term SRCs [8]. Thus, human short- and long-term HSCs can be prospectively separated using adenoviral reporting of hTERT expression [8]. This finding is of particular interest, considering that shortterm (6-week) SRCs have a thus far indistinguishable immunophenotype compared with long-term SRCs that repopulate the bone marrow of secondary NOD/SCID mouse recipients. Hence, this separation strategy will facilitate future studies on the biologic differences between long-term and short-term human candidate HSCs. Our studies also indicated that the utility of dGFP with a short half-life of approximately 2 hours was important for reducing the time delay between the detection of the reporter and the expression of the gene to be reported. This is particularly important and relevant when the gene to be reported is dynamically expressed in a cell-cycle-dependent manner. Moreover, adenoviral reporting of gene expression is most likely to be successful when relatively strong signaling pathway-specific promoters are used (Ja¨ra˚s and Fan, unpublished observation).

Future developments and prospects for adenoviral vector-mediated gene transfer into human primitive hematopoietic progenitor cells Although the fiber retargeting from Ad5 to Ad35 or Ad11 has greatly improved the gene transfer efficiency into human primitive hematopoietic cells, it is currently unclear why a substantial fraction of the CD34þ cells still lacks transgene expression following adenoviral transduction, given that the vast majority of CD34þ cells express high levels of the CD46 receptor [7]. Theoretically, it could either be due to lack of permissiveness in adenoviral uptake and/or intracellular trafficking steps in a certain subpopulation of CD34þ cells or lack of promoter activity in a successfully transduced cell population [4,37]. Future studies will have to address this issue. Proof-of-principle studies from our laboratory and others have demonstrated that adenoviral vectors can be a powerful tool for studying gene regulation and function in primitive hematopoietic cells. We hypothesize that transient expression of ‘‘stem cell genes’’ such as bmi-1 and gfi-1 [61–63]

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in human primitive hematopoietic progenitor cells may allow expansion of HSCs with direct therapeutic applications, such as expansion of cord blood HSCs prior to bone marrow transplantations. Several of the pathways crucial for normal HSC self-renewal have also been implicated in leukemogenesis. Adenoviral overexpression of potential oncogenes could be used to address the direct effects of specific oncogenes in different leukemias. Furthermore, we speculate that adenoviral reporting of leukemia-specific pathways will allow the separation of leukemic stem cells from their normal counterparts, which potentially could have clinical significance if the reporter is replaced by suicide or immune-stimulatory genes. Moreover, we hypothesize that novel subgroups of normal progenitors will be identified by adenoviral vector reporting of gene expression in CD34þ cells, in a similar manner as was described in the hTERT study [8]. In summary, adenoviral vectors have emerged as powerful tools for transient genetic manipulation of primitive hematopoietic cells. We believe that adenoviral vectors have a great potential in facilitating future studies both in basic research and for clinical applications within the field of hematopoiesis and leukemia research.

Acknowledgments These studies were supported by grants from the Swedish Cancer Society, the Swedish Childhood Cancer Society, the Royal Physiographic Society in Lund, the Crafoord Foundation, the Georg Danielsson Foundation, Siv-Inger & Per-Erik Anderssons Minnesfond, the Funds of Lund University Hospital, the Hedvig Foundation, and the Swedish Gene Therapy Program. Xiaolong Fan is a Li Ka Shing Scholar. We thank Anna Edqvist and Dr. Seema Rosqvist for critical reading of the manuscript.

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