Experimental Hematology 2010;38:707–717
Gene transfer into human cord bloodderived CD34þ cells by adeno-associated viral vectors Natascha K. Schuhmanna,*, Ombretta Pozzolib,*, Jessica Sallacha,c, Anke Hubera,c, Daniele Avitabiled, Luca Peraboa, Gunter Rappla,c, Maurizio C. Capogrossid, Michael Halleka,c, Maurizio Pesceb,*, and Hildegard Bu¨ninga,c,* a
Department I of Internal Medicine, University of Cologne, Cologne, Germany; bLaboratorio di Biologia Vascolare e Medicina Rigenerativa, Centro Cardiologico MonzinoIRCCS, Milan, Italy; cCenter for Molecular Medicine Cologne (ZMMK), University of Cologne, Cologne, Germany; d Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’ ImmacolataIRCCS, Rome, Italy (Received 21 January 2009; revised 8 April 2010; accepted 27 April 2010)
Objective. Bone marrowLderived CD34+ cells are currently used in clinical trials in patients with ischemic heart disease. An option to enhance activity of injected progenitors may be offered by genetic engineering of progenitor cells with angiogenic growth factors. Recombinant adeno-associated viral vectors (rAAV) have emerged as a leading gene transfer systems. In contrast to other vector systems in use for genetic engineering of CD34+ cells, rAAV-mediated gene expression does not depend on vector integration. This is relevant for application in regenerative medicine of ischemic tissues, where transient transgene expression is likely sufficient to achieve therapeutic benefits. Materials and Methods. We compared three different human AAV serotypes, packaged as pseudotypes by a helper virus-free production method, for their transduction efficiency in human cord bloodLderived CD34+ cells. We further assessed the impact of vector genome conformation, of avb5 and a5b1 integrin availability and of the transcription-modulating drugs retinoic acid and Trichostatin A on rAAV-mediated human CD34+ cell transduction. Results. We provide, for the first time, evidence that hCD34+ cells can be reproducibly transduced with high efficiency by self-complementary rAAV2 without inducing cytotoxicity or interfering with their differentiation potential. We further show the involvement of a5b1 integrin as a crucial AAV2 internalization receptor and a function for transcriptionmodulating drugs in enhancing rAAV-mediated transgene expression. Conclusion. This study represents a first step toward translation of a combined cellular/ rAAV-based therapy of ischemic disease. Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
Use of bone marrow (BM) and peripheral blood (PB)–derived progenitors represents an interesting therapeutic option for treating patients with peripheral and heart ischemic disease (reviewed in ). In fact, different clinical trials of autologous BM- and PB-derived progenitor administrations have been conducted. The results, although interesting, are still modest in terms of clinical benefits [2–5]. The limited efficacy in patients is due to different causes, such as reduced bioactivity
*Drs. Schuhmann and Pozzoli contributed equally to this work, as did Drs. Pesce and Bu¨ning. Offprint requests to: Hildegard Bu¨ning, Ph.D., University of Cologne, Department I of Internal Medicine, Center for Molecular Medicine Cologne (ZMMK), ZMMK Research Building, Robert-Koch-Str. 21, Cologne 50937, Germany; E-mail: [email protected]
of progenitors obtained by older persons or by subjects with cardiovascular risk factors [6–9], the poor survival and ability to proliferate after transplantation, and the inflammatory response due to ischemia that limits the ability of injected progenitors to successfully engraft . Application of ‘‘enhancement strategies’’ aimed at improving the proliferation, survival, and engraftment ability of progenitor cells into ischemic tissues has become a primary end point in cardiovascular regenerative therapy ; different methods to enhance performance of progenitors to be injected in the ischemic heart and limb have been proposed, such as improvement in cell isolation and storage conditions , adoption of stem cell preconditioning methods in culture with chemokines , or genetic engineering [13–15] using viral vectors.
0301-472X/$ - see front matter. Copyright Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2010.04.016
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In addition, translation of stem cell ‘‘enhancement strategies,’’ such as genetic engineering, requires use of protocols that maximize the safety for patients and lacks the interference with stem cell survival or differentiation potential. Although of extremely valuable significance for an understanding of basic processes underlying regeneration potential by stem cells, most of the studies describing preclinical efficacy of genetic modification of progenitor cells have been conducted using vectors that do not entirely comply to strict requirements of safety. In fact, retroviral or lentiviral vectors provide a potential risk for insertional mutagenesis, while adenoviral vectors elicit strong immune responses in the host, even though they have been employed in clinical trials in humans (reviewed in [16–18]). Recently, adeno-associated viruses (AAV), nonpathogenic members of the parvovirus family, have gained increasing attention as gene delivery vectors. In fact, multiple recombinant AAV (rAAV) serotypes and variants differing in tissue tropism have been applied for ex vivo gene transfer and in preclinical animal models (reviewed in [19,20]). Moreover, O60 clinical trials have been approved, mainly for treatment of inherited diseases and cancer [21–27]. The advantage offered by rAAV consists in a relatively high transgene expression level that is not dependent on stable integration of transgene DNA [19,20]. Based on these considerations and prompted by recent improvements in murine hematopoietic stem cell transduction with rAAV, we evaluated the ability of different human AAV serotypes to transduce CD34þ cells isolated from human (h) cord blood (CB). In the present study, we identified some of the critical steps in rAAV-mediated gene transfer into these cells. In addition, we describe an optimized procedure for transduction of hCD34þ cells using the transcriptionmodulating drugs retinoic acid (RA) and Trichostatin A (TSA). This method is prospectively of translational interest as a novel enhancement strategy for the treatment of heart ischemic disease by using progenitor cells.
Materials and methods Preparation and culture of hCD34þ cells hCD34þ cells were isolated from CB using Ficoll-density gradient purification followed by positive selection (Direct CD34 Progenitor Cell Isolation Kit; Miltenyi Biotech, Bergisch Gladbach, Germany). Cell purity was 80% to 90%, as routinely determined by flow cytometry. In addition, CB-derived hCD34þ cells were purchased from CellSystems (St. Katharinen, Germany), STEMCELL Technology (Vancouver, BC, Canada) and AllCells (Emeryville, CA, USA) and were used where indicated. All cells were cultivated in Stem Span stem cell expansion medium (CellSystems) containing the cytokines interleukin (IL)-3 (20 ng/mL), IL-6 (20 ng/mL), Flt3-ligand (100 ng/mL), and stem cell factor (100 ng/mL) at 37 C in a humidified CO2 (5%) incubator as described previously [28,29]. hCD34þ cells were pre-expanded for 2 to 4 days prior to transduction.
Vector preparation Vectors were prepared by a helper virus-free method. Briefly, HEK293 cells were transfected by the vector plasmids pGFP or pscAAV/enhanced green fluorescent protein (GFP), an AAV helper plasmid coding for the nonstructural proteins of AAV2 and the structural proteins of the respective serotype (pXR2, pXR3, and pXR5) or mutant (pR513A), and the adenoviral helper plasmid pXX6 [30–33]. Forty-eight hours post-transfection, cells were harvested and lysed. Cellular DNA and RNA, as well as remaining plasmid DNA, were removed by Benzonase treatment and cellular debris was removed by centrifugation . Cleared lysate was loaded onto a discontinuous iodixanol density gradient and vectors were harvested from the 40% phase of the gradient . The genomic titer was determined by quantitative polymerase chain reaction  and ranged between 6.6 1010 (rAAV5sc) and 1.1 1012 (rAAV2ss) genomic particles/mL. Transduction of hCD34þ cells and detection of transduction efficiency The 8 104 pre-expanded hCD34þ cells were transduced with the respective amount of genomic particles per cell (GOI) or solvent (mock-transduction). Three days post-transduction, cells were harvested. After a washing step, transgene expression was determined by flow cytometry. In competition studies, cells were transduced in the presence and absence of 971 U heparin (Sigma-Aldrich, St Louis, MO, USA). For studies on transcription activity, hCD34þ cells were transduced in the presence of TSA (25 ng/mL; Sigma-Aldrich) and/or RA (10 mM; Sigma-Aldrich). Colony-forming units endothelial cell differentiation assay After thawing, hCD34þ cells (AllCells) were plated and expanded for 3 days as described. Cells were then incubated with rAAV2sc (at a GOI of 1 104) for 18 hours, followed by an extensive washing in phosphate-buffered saline. After washing, cells were cultured in expansion medium supplemented with cytokines in the presence of TSA (25 ng/mL) and RA (10 mM). As a control, cells were incubated with solvent instead of rAAV2sc and treated as described here. rAAV2sc-transduced and mock-transduced cells were plated for the same time under these conditions. Finally, to promote differentiation into colony-forming units endothelial cell (CFU-EC) cells , rAAV2sc-transduced and mock-transduced cells were plated into fibronectin-coated dishes in M199 medium supplemented with 20% fetal bovine serum . Culture in differentiation medium was performed for 3 days, followed by flow cytometry. To do this, cells were detached from culture plates using a nonenzymatic cell dissociation solution for fluorescence activated cell sorting analysis (Sigma-Aldrich), followed by antibody incubation (see Antibody detection of cell surface molecules). Flow cytometry was performed using a FACSAria (Becton-Dickinson, Franklin Lakes, NJ, USA). Antibody detection of cell surface molecules Cells were stained for 15 minutes with anti-avb5 (MAB1961; Chemicon-Millipore, Billerica, MA, USA) or anti-a5b1 (MAB1999; Chemicon-Millipore) antibody diluted 1:50 in phosphate-buffered saline. After a washing step, cells were incubated with the secondary antibody (goat anti-mouse immunoglobulin Gphycoerythrin [PE]labeled polyclonal antibody; Abcam, Cambridge, MA, USA) at the same dilution. Analysis of CD34, CD133, CD105, and CD31 expression was performed by adding appropriate dilutions of anti CD34-PE (Becton Dickinson), antiCD133-allophycocyanin (APC)
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(Miltenyi Biotech), antiCD105-PE (R&D Systems, Minneapolis, MN, USA), antiCD31-APC (Miltenyi Biotech), or isotype control antibodies for 15 minutes at room temperature prior to flow cytometric analysis. To assess the CFU-EC phenotype, the following antibodies were used: CD105-Alexa700, CD146-APC, CD3-PacificBlue, KDR-PE, CD14-APCH7, CD144-PE, CD48-Alexa700, and CD45PE. These antibodies were diluted and used according to manufacturer’s instructions (BD Pharmingen, San Diego, CA, USA). Statistical analysis Statistical analysis was performed using Graph-Pad Prism 5 statistical software (La Jolla, CA, USA). All experiments were performed at least in triplicate. Statistical comparison was performed by Student’s t-test.
Results rAAV2 is a suitable serotype for transduction of hCD34þ cells Different AAV serotypes are showing considerable promise as gene transfer vectors. Consequently, we aimed to compare different human AAV serotypes for hCD34þ cell transduction. To this aim, we included the following improvements in AAV vector development: pseudopackaging to exclude influences on transduction other than those provided by the viral capsid ; viral vector production in helper virus-free conditions to avoid any assistance of helper virus particles on rAAV transduction ; and packaging of vector genomes as single-stranded ([ss], natural genome conformation) and
double-stranded (self-complementary [sc]) DNA , respectively, to determine the impact of genome conformation on transduction efficiency. To identify the most efficient serotype to proceed with hCD34þ cell transduction, we pre-expanded hCD34þ cells isolated from umbilical CB in hematopoietic stem cell expansion medium supplemented with IL-3, IL-6, Flt3ligand, and stem cell factor [28,29]. The phenotype of these cells was assessed at the end of a 3-day expansion period that revealed expression of CD34 and CD31 markers at relatively high levels (Fig. 1). Pre-expanded cells were transduced with equivalent numbers of vector genomes (105 genomic particles per cell; GOI) of rAAV serotype 2 (rAAV2), rAAV3, and rAAV5, respectively, encoding for the GFP in the two available vector genome conformations. As shown in Figure 2A, rAAV3ss or rAAV5ss did not produce detectable transductions, while rAAV2ss achieved a low transduction efficiency (8% 6 2.5%, n 5 8). In contrast, rAAV with self-complementary vector genome configurations showed a detectable GFP expression for all three serotypes, with a maximal efficiency for rAAV2sc (57.2% 6 2.6%; n 5 8). Exclusion of pseudotransduction by heparin competition Alexander and colleagues reported that CD34þ cells are the subject of pseudotransduction . To exclude this possibility, we performed competition studies using heparin, a soluble analogue of AAV2’s primary receptor heparan sulfate proteoglycan . As shown in Figure 2B, heparin
Figure 1. Phenotype of human (h) CD34þ cells following pre-expansion. Cord bloodderived hCD34þ cells were pre-expanded in hematopoietic stem cell expansion medium supplemented with interleukin (IL)-3, IL-6, Flt3-ligand, and stem cell factor as described in references [28,29]. After 3 days, cells were analyzed by flow cytometry to assess the vitality (7-AAD exclusion test) and the expression of CD34, CD133, CD31, and CD105 markers. Results are shown as mean 6 standard error of mean of three independent experiments with cells from pooled donors (STEMCELLTechnology). FSC 5 forward scatter; SSC 5 side scatter; 7-AAD 5 7-amino actinomycin D.
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Figure 2. Transduction efficiency of adeno-associated viral vector serotypes 2 (rAAV2), rAAV3, and rAAV5 on human (h) CD34þ cells. (A) rAAV-mediated transductions of hCD34þ cells. The 8 104 pre-expanded, cord blood (CB)derived hCD34þ cells were transduced with a genomic particle per cell ratio (GOI) of 105 for 3 hours at 37 C using the indicated serotypes encoding for enhanced green fluorescent protein (GFP) and containing either a single-stranded (black bars) or self-complementary (gray bars) vector genome conformation. Transduction efficiencies were determined by flow cytometry 3 days p.i. (post infection). Values are shown as mean percentage of positive cells 6 standard error of mean. In total, three independent experiments were performed comparing all three serotypes, and for rAAV2, five additional donor samples were analyzed (n 5 8). For each experiment cells were freshly isolated from umbilical CB of the respective donor. (B) Heparin competition assay. hCD34þ cells freshly isolated from cord blood followed by ex vivo preexpansion were transduced with a GOI of 105 for 3 hours at 37 C by self-complementary rAAV2 (rAAV2sc) in the presence or absence of heparin (971 U). Cells were analyzed by flow cytometry 3 days p.i. GFP 5 enhanced green fluorescent protein.
addition dramatically reduced rAAV2sc transduction efficiency from 56.7% 6 5.3% to 1.2% 6 0.7% (n 5 3; p ! 0.0003), thus excluding pseudotransduction. Phenotypic characterization of rAAV2sc-transduced cells To assess a possible impact of rAAV2sc on survival and/or phenotype of hCD34þ cells, a multiparametric analysis of rAAV2sc-transduced and mock-transduced hCD34þ cells was performed by flow cytometry at 3 days posttransduction. To this aim, staining with 7-amino-actinomycin D (7-AAD) was conducted, followed by incubation with antibodies recognizing progenitor cell-specific and endothelial cell-specific antigens. As shown in Figure 3, only a minimal portion (around 2.5%) of AAV-transduced cells was caused to die, i.e., stained positive for 7-AAD. Further gating of 7-AAD/GFPþ cell fraction revealed that the main proportion of rAAV2sc-transduced cells displayed CD34, CD31, or CD31/105 (Fig. 3). Transduction efficiency correlates with availability of a5b1 integrin Previous investigations have postulated that donor-specific differences in the availability of cellular co-receptors are responsible for the high variability in transduction efficiencies in hCD34þ cells . Two integrin co-receptors, avb5 and a5b1, involved in AAV2 internalization have been identified [30,40]; however, a correlation between co-receptor availability and transduction efficiency for hCD34þ cells is still missing. To assess this issue, avb5 and a5b1 integrin availability and rAAV2sc transduction efficiency were analyzed in parallel by flow cytometry (Fig. 4A). As shown in Figure 4B, cells from different hCD34þ cell preparations
were comparably transduced (percentage of GFPþ cells: 61.8% 6 5.7%, n 5 4). These cells expressed high and comparable a5b1 integrin levels, but showed a striking variation in avb5 expression (Fig. 4B). To further explore the function of a5b1 integrin in rAAV2sc transduction, we exploited the a5b1 integrin-binding mutant rAAV2-R513A . We compared the transduction efficiency of this mutant to that of rAAV2sc. In these experiments, we used a GOI of 104 because the mutant could only be packaged with low genomic particle titers. The results showed a significant reduction in the efficiency of R513A compared to rAAV2sc transduction (Fig. 4C), supporting the hypothesis that a5b1 integrin is involved as a co-receptor in rAAV2-mediated transduction of CB-derived hCD34þ cells. Transcription-modulating drugs enhance rAAV-mediated transgene expression The rAAV2sc encodes for GFP controlled by the cytomegalovirus promoter. This promoter contains several functional RA responsive elements . RA modulates gene expression by activating RA receptors that, in turn, associate with a number of co-activators, some of which are members of the histone acetyltransferase (HAT) family. In this context, the histone deacetylase (HDAC) inhibitor TSA is known to enhance the transcriptional enhancing effect of RA . Therefore, we evaluated whether RA and TSA alone or in combination enhance rAAV2sc-mediated transgene expression and thereby further increase our transduction efficiency. We initially treated CB-derived hCD34þ cells with rAAV2sc in the presence of different RA (55,000 nM) and/or TSA
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Figure 3. Phenotype of rAAV2sctransduced hCD34þ cells. Pre-expanded CBderived hCD34þ cells were transduced with a GOI of 105 (n 5 2) or 2.5 105 (n 5 1) for 3 hours at 37 C. After 3 days, cells were analyzed by flow cytometry to assess the vitality (7-AAD exclusion test), the enhanced green fluorescent protein expression and the expression of CD34, CD133, CD31, and CD105 markers. Cells were sequentially gated on the basis of their (i) scatter parameters (forward scatter [FSC], side scatter [SSC]), (ii) their negativity/positivity for 7-AAD, and (iii) their GFP expression.
(3.125500 ng/mL) amounts, to determine the best concentration of either drug. These analyses revealed an optimal concentration of 10 mM for RA and of 25 ng/mL for TSA (not shown). Next, we incubated pre-expanded CB-derived
hCD34þ cells with rAAV2sc and either drug, alone or in combination, and determined the percentage of transgene expressing cells, as well as the mean fluorescence intensity, as a value for transduction efficiency and transcriptional
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Figure 4. avb5 and a5b1 integrins and transduction efficiency. (A), (B) Correlation between availability of avb5 and a5b1 integrins and transduction efficiency. After a 3-day pre-expansion, CBderived hCD34þ cells were assayed for the presence of avb5 and a5b1 integrin displaying cells by flow cytometry [representative example in (A)]. Subsequently, cells were transduced with a GOI of 105 of rAAV2sc for 3 hours at 37 C. Transduction efficiency was determined 3 days p.i. by flow cytometry. (C) Transduction efficiency of integrin non-binding mutant. Following a 3-day pre-expansion, CB-derived hCD34þ cells were transduced with a GOI of 104 for 3 hours at 37 C by rAAV2sc or rAAVsc-R513A (a5b1-integrin non-binding mutant). Transduction efficiency was determined 3 days p.i. by flow cytometry. Results are shown as mean 6 standard error of mean in three independent experiments. GFP 5 enhanced green fluorescent protein.
activity, respectively. As shown in Figure 5, the addition of both drugs in combination revealed an additive effect on the increase in mean fluorescence intensity (Fig. 5A) and the percentage of cells expressing GFP compared to control cells (Fig. 5B). CFU-EC phenotype of rAAV2sc-transduced hCD34þ cells CD34þ cells from BM, CB, and PB are recognized as endothelial progenitor cells (EPCs) capable of differentiating into two alternative phenotypes: the CFU-EC (also called ‘‘early EPCs’’) and the endothelial cell colony-forming cells (also named ‘‘late’’ EPCs) (reviewed in ). Although early CFU-ECs arise from the CD45þ cell fraction in the CD34þ population and endothelial cell colonyforming cells derive from rare clonogenic cells present in the CD34þ/CD45 fraction , both cell types have a potentially therapeutic value to induce formation of blood vessels into ischemic tissues . Here, we focused exclusively on the impact of rAAV2sc-mediated transduction on
the differentiation potential of CB-derived hCD34þ cells into cells having a CFU-EC phenotype for the reason that cells capable of endothelial cell colony-forming are very rare in the BM or CB mononuclear fraction and in the CD34þ cell population, and that their colony-forming frequency is very low (around 110 colonies/108 cells ) and, therefore, not compatible with the amount of CD34þ cells that could be transduced per experiment. CFU-ECs have been reported to have a mixed myeloid/ lymphoid/endothelial phenotype (for references see [45– 49]). To assess whether transduction with rAAV2sc induces changes in CFU-EC phenotype, CB-derived hCD34þ cells were first mock- or rAAV2sc-transduced followed by culture under conditions reported to favor the formation of CFU-EC colonies and multiparametric analysis in flow cytometry to recognize markers typically expressed in these cells. To this aim, we used antibodies raised against CD3, CD45, CD48, CD105, CD144, CD146, and vascular endothelial growth factor receptor2/KDR and analyzed the expression of these markers in the total (mock-transduced)
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Figure 5. Enhanced rAAV2scmediated transgene expression by addition of transcription-modulating drugs. The 8 104 pre-expanded CBderived hCD34þ cells were treated with 25 ng/mL trichostatin A (TSA) and/or 10 mM retinoic acid (RA) at the time of transduction, and transduced with rAAV2sc at a GOI of 105. In parallel, cells were transduced with the same amount of vectors in the absence of the drugs (only solvent). Three days p.i. transgene expression was assessed by flow cytometry. Depicted are the mean fluorescence intensity [MFI; (A) and the percentages of transgene (GFP)-expressing cells (B). Results are shown as mean 6 standard error of mean; n 5 3.
and GFPþ gated (rAAV2sc-transduced) cells. The results (Fig. 6) showed that, with the exception of minor differences in the percentage of cells expressing the pan-hematopoietic marker CD45 and the endothelial marker KDR, rAAV2sctransduced were indistinguishable from mock-treated hCD34þ-derived CFU-ECs.
Discussion Human CD34þ cells are of cardiovascular therapeutic interest for their ability to induce, directly or indirectly, formation of new blood vessels into tissues affected by ischemia (reviewed in [1,50–52]) and are currently used in clinical trials to treat patients with myocardial ischemia [53–55]. Pharmaceuticals [56–60], gene transfer [15,61], and culture preconditioning [10–12] are strategies that have been used to enhance EPC regeneration potential (also reviewed in ). Although use of culture preconditioning is aimed at restoring the innate hEPC functions that are lost due to cardiovascular risk factor , application of genetic engineering strategies is instrumental to use these cells as delivery tools of pro-angiogenic factors into ischemic tissues . So far, hEPC gene transfer has been attempted using first-generation adenovirus type 5based vectors to overexpress angiogenic factors, such as vascular endothelial growth factor [15,61,63] in human PB-derived EPCs. Alternatively, it has been performed using lentiviral vectors . Adenovirus type 5based vectors have a limited ability to transduce selected hEPCs populations, such as hCD34þ cells (our unpublished observations), and requires use of vectors containing a modified capsid, such as the mixed 5/35 adenovirus serotype . In addition, they potentially elicit strong immune responses in the hosts and have a high cytotoxicity.
Lenti- and retroviral-mediated gene transfer, although used already in therapy for correction of childhood immunological diseases (retroviral vectors) and in the treatment of HIV infection (lentiviral vectors), are not devoid of risks, as they can cause insertional mutagenesis due to random integration into the host-cell genome [16,17]. It turns out that there is still no reliable and optimized system to overexpress therapeutic genes in selected progenitor cell populations to induce neovascularization, which is highly efficient, does not cause massive death of transduced cells, does not induce host immune responses, and has a low incidence of random integration. Given the growing interest in rAAV as an alternative gene delivery method in clinical trials, we decided to assess the feasibility of rAAV transduction in hCD34þ cells as a step towards clinical translation of a combined cell/gene therapy approach to combat the consequences of ischemic disease. rAAV2sc mediates reproducible transgene expression by hCD34þ cells rAAV has been applied in clinical trials of acquired and inherited diseases and, recently, evidence for clinical efficacy was reported for Parkinson disease, lipoprotein lipase deficiency, and Leber congenital amaurosis [21–27]. Besides serotype 2, alternate AAV serotypes have entered human gene therapy, owing to the superior transduction efficiency of clinical relevant cell types, such as muscle or liver parenchymal cells (reviewed in ). In the present study, we compared the efficiency of three human AAV serotypes (rAAV2, rAAV3, and rAAV5) for their ability to transduce human CB-derived CD34þ cells. rAAV3 is 82% homolog to AAV2  and binds to the same receptor ; however, it has a specific tropism for
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Figure 6. Phenotype of colony-forming units endothelial cell (CFU-EC). To assess differentiation potential of mock and rAAV2sctransduced CBderived hCD34þ cells, they were cultured under conditions promoting differentiation into CFU-ECs. After 3 days, cells were analyzed for expression of CD3, CD45, CD48, CD105, CD144, CD146, and vascular endothelial growth factor receptor 2 (VEGFR2)/KDR. Mock-transduced cells were gated on the total cellular population (A), while rAAV2sctransduced cells were gated on the basis of their enhanced green fluorescent protein positivity (B). Results are shown as mean 6 standard error of mean (C); n 5 3; *p ! 0.05 in paired Student’s t-test.
hematopoietic cells . rAAV5 is the most divergent of the AAV serotypes ; it uses sialic acid as attachment molecule  and platelet-derived growth factor receptora  as a co-receptor. In contrast to murine hematopoietic stem cells (mHSCs), which have been more efficiently transduced using rAAV1ss compared to rAAV2ss , and to human PB-derived progenitor cells, for which AAV6ss was superior in comparison to AAV2ss , AAV2ss clearly outperformed rAAV3ss and rAAV5ss in transduction of CB-derived hCD34þ cells (Fig. 2). However, even with this serotype, we transduced not more than 11% of the cells when a single-stranded vector genome was delivered (Fig. 2). A single-stranded vector genome corresponds to the naturally occurring genome conformation with a coding capacity of approximately 5 kb. It has to be converted into double-stranded DNA prior to transgene expression . This step can be circumvented using self-complementary vector genomes , which are packaged as inverted repeat genomes that fold into a double-stranded DNA without the need for DNA synthesis. A significant increase (O7-fold) in the transduction efficiency for all three serotypes was obtained by using rAAV with self-complementary genomes (Fig. 2); this result reveals that second-strand DNA is a major critical step in CB-derived hCD34þ cell transduction (Fig. 2) and is in line with reports on mHSCs and PB-derived progenitor cells [72,74,75].
The discrepancy observed in the transduction efficiency of rAAVss reported here compared to previous studies may result from the difference in the vector preparation protocols. Here, we used a helper virus-free rAAV packaging protocol, which is in contrast to earlier studies in which vector production protocols included the presence of helper viruses [39,76–78], which, in natural infections, is known to support single-to-double-strand conversion of single-stranded AAV genomes [79,80] by, for example, E4orf6 protein. This makes it likely that successful transduction of hCD34þ cells with rAAV with single-stranded vector genomes reported in previous reports was due, at least in part, to helper virus assistance in single- to doublestrand conversion. Involvement of a5b1 integrin in successful transduction by rAAV2sc Ponnazhagan and colleagues have reported donor-specific variations in the susceptibility of BM-derived hCD34þ cells to be transduced by rAAV2 . In that study, only half of their donor samples were transduced with an efficiency ranging from 15% to 80%. By contrast, Nathwani and colleagues  reported more constant rAAV2ss transduction efficiency levels in hCD34þ and hCD34þCD38 cells. Although the transduction obtained in that report was 2.9fold higher compared to our results for rAAV2ss , it
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has to be noted that in that study 1,000-fold higher vector particle per cell ratios (GOI w108 vs GOI of 105) were used. According to the currently accepted models, AAV enter cells by receptor-mediated endocytosis. Therefore, differences in transduction efficiencies can result from variable availability of internalization receptors such as, for example, avb5 and a5b1 integrins [30,40,82]. The results obtained in the present study suggest that a5b1, rather than avb5, integrin determines the susceptibility of CB-derived hCD34þ cells to rAAV transduction because we observed relatively high and constant a5b1 levels in pre-expanded cells that were comparably transduced by rAAV2sc, while in the same cells the availability of avb5 was highly variable from cell-to-cell preparation (Fig. 4B). In addition, a mutant vector rAAVsc-R513A, which is deficient for a5b1-integrin binding , was 5.6-fold less efficient than rAAV2sc in hCD34þ cell transduction (Fig. 4C).
showed that CFU-EC differentiation of hCD34þ cells (Fig. 6) was not affected by transduction with rAAV2sc.
Addition of RA and TSA improves rAAV2sc transgene activity Our results show that transgene activity achieved in preexpanded CB-derived hCD34þ cells by rAAV2sc was further enhanced by the addition of TSA and RA. This is consistent with a report by Gaetano and colleagues showing a synergistic effect of both drugs in adenoviral vectormediated gene transfer in cell culture and in vivo . The increase likely resulted from the combined recruitment of trans-acting RA receptors to RA responsive elements (cis-regulatory elements present in the cytomegalovirus promoter), and the inhibition of class I and II HDAC activity that is known to repress trans-activation by modifying chromatin conformation. In addition, as HDAC inhibitors influence the balance between the activity of HDACs and HATs, enhanced activity of HATs, which act as co-activators of RA receptors, may also promote gene transcription from RA responsive elementcontaining promoters . We hypothesize that enhancement of cytomegalovirus promoter activity by combinatorial effect of TSA and RA likely resulted in the increase in transduction efficiency (Fig. 5). In fact, as also suggested by results on tumor necrosis factora enhancement of AAV transgene expression activity , increase in GFP expression likely resulted in an elevation of fluorescence level above the detection threshold in a significantly higher number of cells.
This work was supported by the European Union SC&CR (LSHB-CT-2004-502988) (M.H., M.C.C., and M.P.), (LSHBCT-2005-512102) (M.H., M.C.C., and M.P.), and Thercord (LSHB_CT_2005-018817) (M.P.), from the German Research Foundation (SPP 1230 and SFB 670 [H.B. and M.H.], HA 1680/ 8 [M.H.]) and the Center of Molecular Medicine Cologne (University of Cologne, Cologne, Germany) (H.B. and M.H.). We thank Prof. Richard Jude Samulski (University of North Carolina at Chapel Hill, NC, USA) for providing the plasmids pXX6, pXR2, pXR3, and pXR5. Furthermore, we thank Michele Cadau (Centro Cardiologico Monzino, Milan) for technical assistance. The authors thank Prof. Andrea Biondi (Clinica Pediatrica, Universita` degli Studi di Milano Bicocca, Monza, Italy), Dr. Daniela Longoni (Centro Ricerche Tettamanti), Dr. Domenica Mammoliti (Ospedale di Melzo, Milan, Italy), for kind assistance in collection of human CB, and Tobias Riet and Dr. Markus Chmieleweski (University of Cologne, Cologne, Germany) for kind assistance in flow cytometry.
Concluding remarks Our results show that transduction of CB-derived hCD34þ cells is feasible using rAAV2sc and transcriptionally active drugs, such as RA and TSA. Besides identifying the critical importance of factors involved in efficient transduction of these cells by AAV2, our results provide an alternative option to introduce transgenes into cells having EPC activity for clinical settings. Future studies are necessary, however, to assess the therapeutic relevance of rAAV2sctransduced hCD34þ cells using suitable animal models of myocardial and/or peripheral ischemia, and to validate their safety by appropriate genetic investigations.
rAAV transduction does not interfere with CD34þ cell differentiation Recently, Maina and colleagues transduced mHSCs with rAAVs, followed by transplantation into lethally irradiated syngeneic recipient mice . Even at 6 months after transplantation, transgene expression was detectabledin line with the promoter specificitydin cells of the erythroid lineage . This revealed that rAAV transduction did not affect mHSC engraftment ability and allowed long-term transgene expression. In keeping with these observations, our results
Conflict of Interest Disclosure The authors reported no conflict of interest.
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