Functionally Distinct Subpopulations of Cord Blood CD34 Cells Are

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doi:10.1016/j.ymthe.2003.12.014

Functionally Distinct Subpopulations of Cord Blood CD34+ Cells Are Transduced by Adenoviral Vectors with Serotype 5 or 35 Tropism Marcus Nilsson,1,* Stefan Karlsson,1 and Xiaolong Fan1,2 2

1 Department of Molecular Medicine and Gene Therapy and Department of Immunology, Lund University, SE-22184 Lund, Sweden

*To whom correspondence and reprint requests should be addressed. Fax: 0046 46 222 0568. E-mail: [email protected].

Adenovirus serotype 5 (Ad5)-based vectors can be retargeted with fiber receptor specificity of serotype 35 adenovirus (Ad5F35) and thereby bypass the paucity of the coxsackie and adenovirus receptor (CAR) on hematopoietic cells by utilizing CD46 as cellular receptor. The gene transfer efficiency into NOD/SCID repopulating cells by an Ad5F35-GFP vector was investigated in comparison with its corresponding Ad5-GFP vector. Cord blood CD34+ cells were transduced following overnight culture under serum-free conditions supported by early acting cytokines. In agreement with previous findings, the Ad5F35-GFP vector showed significant superiority to the Ad5-GFP vector in gene transfer into cells with primitive immunophenotype. However, the Ad5F35-GFP vector allowed efficient gene transfer into both dividing and nondividing CD34+ cells, whereas the Ad5-GFP vector preferentially allowed gene transfer into dividing cells expressing lower levels of CD34 antigen, which correlated with high levels of CAR expression. The sorted GFP+ cells following Ad5F35-GFP transduction at relatively low multiplicity of infection consistently reconstituted the NOD/SCID mouse bone marrow with multilineage differentiation. In contrast, the GFP+ cells following Ad5-GFP transduction were nearly devoid of reconstitution capacity. Thus, Ad5F35 vectors encoding functional genes can facilitate transient genetic manipulation of human NOD/SCID repopulating cells. Key Words: NOD/SCID repopulating cells, recombinant adenoviral vector, fiber retargeting, gene transfer

INTRODUCTION Hematopoietic stem cells (HSCs) are capable of self-renewal and multilineage differentiation into all mature blood cells [1]. This process is controlled by a stringent regulation of gene expression in HSCs, such that at any given time point, only a small fraction of HSCs are proliferating and responsible for the generation of multilineage mature blood cells, whereas the majority of the HSCs are in quiescence [2]. Dysregulated proliferation and differentiation of HSCs and primitive hematopoietic progenitor cells can cause leukemia [3]. The molecular mechanisms underlying these processes are of intense interest for many research groups. Since adenoviral vectors normally do not integrate into the host cell genome, successfully delivered genes via adenoviral vectors can be transiently expressed in HSCs. This feature can be desirable for mechanistic studies and ex vivo manipulation of

MOLECULAR THERAPY Vol. 9, No. 3, March 2004 Copyright B The American Society of Gene Therapy 1525-0016/$30.00

HSCs when a persistent gene expression followed by integrating viral vector-mediated gene transfer is not suitable. For adenovirus serotype 5 (Ad5)-based vectors, adenoviral infection of host cells involves two functionally distinct steps. Fiber-mediated attachment to the coxsackie and adenovirus receptor (CAR) is followed by viral internalization via the interaction between the penton base and the av integrins [4,5]. Human primitive hematopoietic cells do not express the CAR and the av integrins at levels allowing for efficient Ad5 vector-mediated gene transfer [6 – 8]. To achieve gene transfer into a substantial fraction of human primitive hematopoietic cells by Ad5based vectors, strategies including prolonged incubation of CD34+ cells with high multiplicities of infection (m.o.i.) of vectors, formation of virus – polycation complexes prior to infection, and cytokine stimulation of CD34+ cells have been explored [9 – 18]. As a proof of principle, our group has previously shown that NOD/

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SCID repopulating cells can be transduced by high m.o.i. of Ad5-based vector with support of the polyamidoamine dendrimer reagent SuperFect [11]. However, the efficacy of Ad5-based vector-mediated transient gene delivery into primitive hematopoietic cells is not sufficient for efficient manipulation of these cells. The Ad5-based vectors can be retargeted by modification of the fiber gene. There are 51 human adenovirus serotypes, which can be divided into species A to F [19]. Different species show distinct tropism likely due to their fiber receptor specificity. The fiber proteins from species A, C, D, E, and F bind to CAR [20], whereas the fiber proteins from species B1 and B2 adenoviruses bind to another cellular receptor(s) [21]. The fiber proteins from B2 species adenoviruses (e.g., Ad11, Ad35) can bind ubiquitously expressed membrane cofactor protein CD46 as a cellular receptor and thereby allow CAR-independent infection of target cells [22,23]. The tropism of Ad5 (species C)-based vectors can be retargeted to that of Ad35. By replacing the entire fiber gene or the knob part in the Ad5 vector genome with the corresponding part of the fiber gene from Ad35, Ad5F35 vectors have been developed [7,24 – 27]. Ad5F35 vectors and Ad35-based vectors show efficient gene transfer into hematopoietic cells with primitive immunophenotype, including the CD34+ cells and primitive progenitor cells from the side population [7,25,28,29]. Since CD34+ hematopoietic progenitor cells and side-population cells are rather heterogeneous in their primitive functionality [30,31], it is unclear whether the Ad5F35 vectors allow gene transfer into NOD/SCID repopulating cells. In this report, we demonstrate that by using a short transduction protocol in the absence of polycationic reagents, an Ad5F35-GFP vector allowed superior gene transfer efficiency into nondividing cord blood primitive progenitor cells. Furthermore, sorted GFP-expressing cells following the Ad5F35GFP vector transduction contained NOD/SCID repopulating cells with multilineage repopulating capacity in NOD/ SCID mice. In contrast, the corresponding unmodified Ad5-GFP vector preferentially allowed gene delivery into dividing CD34+ progenitor cells with nearly undetectable repopulating capacity in NOD/SCID mice. Therefore, Ad5F35 vectors encoding functional genes will potentially facilitate transient genetic manipulation of candidate HSCs.

doi:10.1016/j.ymthe.2003.12.014

the human candidate HSCs, successfully delivered genes need to be under the control of promoters supported by the transcription factor profile in these cells. Our previous studies have shown that the expression cassette with the Pgk1 gene promoter and h-globin gene IVS 2 and polyadenylation signal allows high levels of transgene expression in primitive hematopoietic cells [10,11]. We therefore generated the Ad5F35-GFP vector with the GFP gene under the control of the Pgk1 gene promoter and the h-globin gene IVS 2 and polyadenylation signal (Fig. 1). We compared the Ad5F35-GFP vector-mediated gene transfer efficiency into primitive hematopoietic progenitor cells with that of the Ad5-GFP vector, in which the expression of the GFP gene is also under the control of the Pgk1 promoter and h-globin gene IVS 2 and polyadenylation signal. Since the efficiency of adenoviral vector transduction into human primitive hematopoietic cells can be dependent on the cytokine support [15,18], we cultured cord blood CD34+ cells under serum-free conditions with megakaryocyte growth and development factor (MGDF), stem cell factor (SCF), and Flt3 ligand to induce proliferation of the primitive progenitor cells or with MGDF alone to support survival and minimal proliferation of the primitive progenitor cells [32]. We infected the cells with the Ad5-GFP and the Ad5F35GFP vectors for 3 h and assessed GFP expression 48 h posttransduction. We compared gene transfer efficiency into the cord blood CD34+ cells in the context of fiber receptor specificity and cytokine stimulation. Previous reports indicated that adenoviral vector transduction at m.o.i. of 500 or higher inhibited proliferation of primitive hematopoietic cells [17,18,33]. We observed no obvious difference in cell proliferation and survival between the vector-transduced and the mock-transduced CD34+ cells for both the Ad5-GFP and the Ad5F35-GFP vector in our study. The patterns of GFP expression in the CD34+ cells following adenoviral vector transduction under proliferating conditions are depicted in Figs. 2A and 2B. Distinct populations of GFP-expressing cells were found in the CD34+ cells following the Ad5-GFP or Ad5F35-GFP vector transduction. Compared with the

RESULTS Efficient Gene Transfer into Cord Blood CD34+ Cells by the Fiber-Retargeted Ad5F35-GFP Vector Gene transfer efficiency into human hematopoietic cells with primitive immunophenotype can be significantly improved by using the Ad5-based vectors with Ad35 tropism [7,25,28] or with an Ad35-based vector [29], as these vectors utilize CD46 as a cellular receptor [22,23]. For direct assessment of the gene transfer efficiency into

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FIG. 1. Adenoviral vectors used in this study. Ad5-GFP and Ad5F35-GFP vectors were generated previously [11,27]. In both vectors, the GFP gene is under the control of the Pgk1 promoter and the h-globin gene IVS 2 and polyadenylation signal. (A) The Ad5-GFP vector harbors the unmodified fiber gene. (B) The Ad5F35-GFP vector harbors a modified fiber gene encoding the tail domain of the Ad5 fiber and the shaft and knob domains of the Ad35 fiber.

MOLECULAR THERAPY Vol. 9, No. 3, March 2004 Copyright B The American Society of Gene Therapy

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FIG. 2. Superior gene transfer efficiency into hematopoietic cells with primitive immunophenotype by the Ad5F35-GFP vector. (A) Cord blood CD34+ cells were cultured in serum-free medium supplemented with MGDF, SCF, and Flt3 ligand and transduced with the Ad5-GFP and Ad5F35-GFP vectors at indicated m.o.i. as described under Materials and Methods. Forty-eight hours posttransduction, cells were analyzed for CD34 antigen and GFP expression. The pattern of the GFP expression in CD34+ cells from one representative experiment is shown. The percentage and fluorescence mean intensity of the GFP-expressing cells are shown in each histogram. (B) The percentages and standard deviations of the GFP-expressing cells in CD34+ cells from four independent experiments are summarized. Compared with the Ad5-GFP vector (white bars), the Ad5F35-GFP vector (black bars) transduced significantly higher percentages of cord blood CD34+ cells at all m.o.i. tested (***P < 0.001, t test). (C) Duration of GFP expression following the Ad5-GFP and the Ad5F35-GFP vector transduction in proliferating CD34+ cells. Cord blood CD34+ cells were cultured and transduced with the Ad5-GFP (thin line) and the Ad5F35-GFP (bold line) vector at an m.o.i. of 100 as described for (A) and (B). The average percentages of GFP-expressing cells and their standard deviations in CD34+ cells at the indicated days following vector transduction from three independent experiments are shown.

Ad5-GFP vector, two- to threefold higher percentages of the CD34+ cells were expressing GFP following the Ad5F35-GFP vector transduction. Furthermore, the levels


of GFP expression (the mean intensity of the GFP fluorescence signal) were three- to fourfold higher in cells transduced by the Ad5F35-GFP vector. At an m.o.i. of 20,

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ARTICLE 33 F 6.0% of the CD34+ cells were expressing GFP following transduction with the Ad5F35-GFP, and the GFP-expressing cells increased to 43 F 2.0 and 58 F 3.0% at m.o.i. of 100 and 500, respectively. We observed no proportional increase in the percentages of GFP-expressing cells at higher m.o.i. with either vector, indicating that only a specific subset of CD34+ cells was permissive to infection or only a subset of successfully transduced CD34+ cells allowed high levels of GFP expression. Under the survival condition (MGDF alone), similar patterns of GFP expression in the CD34+ cells were observed (see Fig. 4B and Table 2). Therefore, the Ad5F35-GFP vector allowed superior gene transfer efficiency into cord blood CD34+ cells under both proliferating and survival cytokine conditions.

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To assess the duration of gene expression following the Ad5-GFP and the Ad5F35-GFP vector transduction in CD34+ cells, we transduced CD34+ cells cultured under proliferating conditions at an m.o.i. of 100 and measured the percentage of GFP-expressing cells every second day. As shown in Fig. 2C, relatively high percentages of the GFP-expressing CD34+ cells were observed between day 2 and day 8 following the Ad5F35-GFP vector transduction compared with the Ad5-GFP vector. However, compared with the Ad5-GFP vector, the Ad5F35-GFP vector did not prolong the duration of GFP expression in these cells; no GFPexpressing cells could be detected at day 14 of the culture. During the culture period the net expansion of the cells was on average 50-fold.

FIG. 3. Ubiquitous expression of CD46 and inverse correlation between CAR expression and CD34 antigen expression in CD34+ cells. Mock-transduced cells as cultured for Fig. 2 were assessed for CAR and CD46 expression at the days of transduction (day 1) and GFP expression analysis (day 3). Data shown are from one representative experiment of three performed. Gates on CD34+/low (R1 R2) and CD34high (R1 R3) cells were made as described under Materials and Methods. The CAR expression was increased in cultured cells gradually losing the CD34 antigen expression, while CD46 expression was observed on all cells under tested conditions. In each histogram, the thin line represents the control staining and the bold line represents the CAR or CD46 staining.

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TABLE 1: Percentages of CAR-expressing cells in subpopulations of CD34+ cells under survival and proliferating conditions Condition

Day 1 +

CD34 Survival Proliferating

6.0 F 1.4 4.6 F 1.4

+/low

Day 3 high

CD34

CD34

9.5 F 2.1 10.6 F 0.6

3.1 F 0.1 2.5 F 1.3

+

CD34

CD34+/low

CD34high

13.5 F 0.7 24 F 3.6

18.5 F 0.7 39 F 7.1

10.5 F 6.3 11 F 2.0

Mock-transduced cells were analyzed for CAR expression as described under Materials and Methods and for Fig. 3. Percentages and standard deviations of CAR-positive cells in bulk CD34+, CD34+/low, and CD34high cells at the time of transduction and GFP expression analysis from three independent experiments are shown.

Ubiquitous Expression of CD46 but Inverse Correlation between CAR Expression and CD34 Expression in Cord Blood CD34+ Cells As the Ad5-GFP vector requires CAR expression for infection [4] and the Ad5F35-GFP vector utilizes CD46 as a cellular receptor [22,23], we assessed mock-transduced

cord blood CD34+ cells for CAR and CD46 expression on the days of vector infection (day 1 of culture) and GFP expression analysis (day 3 of culture). As shown in Fig. 3 and Table 1, all cord blood CD34+ cells expressed CD46; however, the CAR expression appeared to be inversely correlated with the CD34 antigen expression. Under

FIG. 4. Superior gene transfer efficiency into nondividing cord blood CD34+ cells by the Ad5F35-GFP vector. (A) PKH26 profiles of cord blood CD34+ cells cultured under proliferating or survival conditions for 60 h. Cells fixed immediately after PKH26 staining were used as controls. The gates defining the dividing and the nondividing cells are indicated. (B) Cord blood CD34+ cells were stained with the membrane dye PKH26, cultured, and transduced with the Ad5-GFP and the Ad5F35-GFP vectors under survival conditions as described under Materials and Methods. The percentages of GFP-expressing cells among dividing or nondividing CD34+ cells as defined in (A) are shown in each dot plot. Data shown are from one representative experiment of three performed.


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proliferating conditions, on day 1 of the culture, a majority of the cells expressed high levels of CD34+ antigen and had barely detectable levels of CAR expression (2.5 F 1.3%); however, a small fraction of CAR-positive cells (10.6 F 0.6%) were found in the CD34+/low cells. During the culture, a fraction of the CD34+ cells differentiated and gradually lost the CD34 antigen expression. On day 3 of the culture, CAR-positive cells were predominantly found in the CD34+/low cells (39 F 7.1%); the frequency and levels of CAR expression in the CD34high cells were remarkably lower (11 F 2.0%). These data are in agreement with previous findings that CAR expression was observed in more differentiated CD34 bone marrow cells and at low levels on immature hematopoietic cells [6 – 8,34]. The Fiber-Retargeted Ad5F35-GFP Vector Exhibited Superior Gene Transfer to Nondividing Cord Blood CD34+ Progenitor Cells Primitive hematopoietic cells at relative quiescence have been demonstrated to be highly enriched for cells with stem cell function as assessed in in vitro long-term culture-initiating cell culture and in vivo NOD-SCID repopulating assays [2,35 – 40]. Primitive hematopoietic cells in active cell cycle progression are associated with loss of stem cell functionality [40]. To investigate the potential of the Ad5F35-GFP vector-mediated gene transfer into nondividing primitive progenitor cells, we performed a cell division tracking study. We stained cord blood CD34+ cells with PKH26 prior to the initiation of culture. As cells divide, the membrane-bound PKH26 molecules are evenly distributed between the daughter cells. Therefore, gene transfer efficiency can be assessed in the context of cell proliferation. We transduced PKH26-stained cells cultured under survival conditions with the Ad5-GFP and the Ad5F35-GFP vectors at m.o.i. of 20, 100, and 500. As shown in Fig. 4A, dividing and nondividing cell populations were defined based on the PKH26 intensity. The nondividing cells showed threefold higher levels of CD34 expression compared with the dividing cells, indicating a higher degree of primitiveness (Fig. 4B). For the Ad5-GFP vector, low percentages of nondividing CD34+ cells were transduced. At an m.o.i. of 500, only 5.6 F 0.3% of the nondividing CD34+ cells expressed the GFP gene, while 37 F 4.9% of the dividing CD34+ cells expressed the GFP gene, indicating that the majority of the Ad5-GFP vector-transduced CD34+ cells were at or had passed through cell cycle progression (Table 2). The Ad5F35GFP vector mediated significantly higher gene transfer into both dividing and nondividing CD34+ cell populations. Nearly equal percentages of the dividing and nondividing CD34+ cells were expressing GFP following the Ad5F35-GFP vector transduction at m.o.i. between 100 and 500. Importantly, 49 F 9.9% of the nondividing CD34+ cells expressed high levels of GFP at an m.o.i.

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TABLE 2: Percentages of GFP-expressing cells in dividing and nondividing CD34+ cells following transduction with the Ad5-GFP or the Ad5F35-GFP vector Vector

m.o.i.

Survival condition CD34+ bulk

Ad5-GFP

Ad5F35-GFP

20 100 500 20 100 500

2.3 7.3 18 27 33 49

F F F F F F

0.2 0.1 1.3 6.7 4.0 5.6

CD34+ nondividing 0.5 1.8 5.6 19 33 49

F F F F F F

0.1 0.3 0.5 3.5 8.5 9.9

CD34+ dividing 4.8 16 37 41 38 56

F F F F F F

0.4 0.2 4.9 17 9.9 11

Cord blood CD34+ cells stained with PKH26 were cultured under survival conditions and transduced with the Ad5-GFP or the Ad5F35-GFP vector at the indicated m.o.i., and the GFP expression in dividing and nondividing CD34+ cells was analyzed as described under Materials and Methods and for Fig. 4. Data shown are the mean percentages and standard deviations of the GFP-expressing cells in different subpopulations from three independent experiments.

of 500. Of note, when the CD34+ cells were transduced with the Ad5F35-GFP vector at an m.o.i. of 20, only 19 F 3.5% of the nondividing CD34+ cells were expressing GFP compared with about two times more cells expressing GFP in the dividing CD34+ cell population, suggesting that dividing cells are favored by the Ad5F35-GFP vector; however, this requirement can be overcome with increased m.o.i. Functionally Distinct Populations of Cord Blood CD34+ Cells Are Transduced by the Ad5-GFP and the Ad5F35-GFP Vector The results presented above showed that the Ad5F35GFP vector mediated efficient gene transfer into both dividing and nondividing CD34+ cells, whereas the Ad5-GFP vector can successfully transduce a substantial fraction of dividing CD34+ cells. These data indicate that the Ad5-GFP and the Ad5F35-GFP vectors may transduce functionally distinct CD34+ cells. To investigate whether both vectors allowed gene transfer into NOD/SCID repopulating cells, we compared directly the NOD/SCID mouse repopulating capacity of the GFP-expressing cells following vector transduction at m.o.i. between 100 and 200 under survival conditions. We sorted the GFP-expressing cells 40 h posttransduction. We transplanted limited numbers (30,000 or 50,000) of the sorted GFP+ cells into each animal via tail vein injection and assessed human cell reconstitution at 6 weeks posttransplantation by flow cytometric analysis of human CD45+ cells in cell suspensions of mouse bone marrow. The GFP-expressing cells following the Ad5F35-GFP vector transduction reconstituted six of seven animals with both lymphoid and myeloid contributions (Figs. 5A and 5B); human primitive progenitor cells expressing the CD34 antigen were consistently observed in the mice with human cell reconstitution. In contrast, the same numbers of


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FIG. 5. Distinct reconstitution capacities of the Ad5GFP and the Ad5F35-GFP vector-transduced cord blood progenitor cells in the NOD/SCID repopulating assay. (A) Engraftment of hu-CD45+ cells in NOD/ SCID mouse bone marrow. The sorted GFP-expressing cells following the Ad5-GFP or the Ad5F35-GFP vector transduction at m.o.i. of 100 or 200 were transplanted together with irradiated accessory cells as described under Materials and Methods. The percentages of human CD45-positive cells in mouse bone marrow cell suspensions at 6 weeks posttransplantation are shown. Symbols indicate different cell doses transplanted in each animal: x for 30,000, n for 50,000, and E for 100,000 cells. Data shown are the sums of two independent experiments. (B) Engraftment and multilineage differentiation of the Ad5F35-GFP vectortransduced NOD/SCID repopulating cells. Data shown are from one representative mouse transplanted with 50,000 GFP + cells following the Ad5F35-GFP vector transduction.

the GFP+ cells following the Ad5-GFP vector transduction showed no or barely detectable levels of reconstitution in seven of seven animals. Comparable numbers of mock-transduced cells were also transplanted, resulting in reconstitution levels similar to those of the mice transplanted with the GFP+ cells following Ad5F35-GFP transduction. These data suggest that under our conditions, Ad5F35-GFP allowed efficient gene transfer into NOD/SCID repopulating cells,


whereas the Ad5-GFP vector allowed gene transfer mainly into progenitor cells with no or very low stem cell function. Primitive hematopoietic cells transduced with the adenoviral vectors have been implied to have diminished functionality due to transduction-related toxicity [7,18]. We therefore compared the repopulating capacity in NOD/SCID mice between sorted CD34 + GFP + and CD34+GFP cells following the Ad5F35-GFP vector trans-

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TABLE 3: The CD34+GFP+ cells following the Ad5F35-GFP vector transduction are not compromised in NOD/SCID mice reconstitution capacity Mouse

Transplanted cells

Chim-erism CD45 (%)

% CD45-positive cells expressing CD15/ CD33

N.C. 1-1-1 1-1-2 1-1-3 1-2-1 1-2-2 1-2-3 1-2-4 1-2-5 N.C. 2-1-1 2-1-2 2-1-3 2-1-4 2-1-5 2-2-1 2-2-2 2-2-3

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

— 105 105 105 105 105 105 105 105 — 105 105 105 105 105 105 105 105

GFP+ GFP+ GFP+ GFP GFP GFP GFP GFP GFP+ GFP+ GFP+ GFP+ GFP+ GFP GFP GFP

0 9.0 14 2.6 15 3.9 18 7.5 0 0 3.9 2.4 9.3 17 2.6 5.9 9.2 34

0 64 79 41 69 67 64 94 — 0 33 37 46 30 29 31 40 40

Number of colonies

CD19

CFU-GM

0 38 19 55 17 31 32 6.5 — 0 71 63 54 73 71 69 60 60

1.3 30 21 7.8 84 6.0 31 21 0.6 0.8 18 9.0 30 55 17 29 99 79

F F F F F F F F

0.6 6.4 6.2 2.7 22 4.1 14 11

F F F F F F F F

3.8 3.0 6.7 6.5 4.8 5.8 4.2 3.9

BFU-E 0 11 5.2 1.5 14 0 6.0 10 0 0 3.6 0.8 3.4 18 5.2 7.0 48 25

F 4.0 F 1.8 F 6.7 F 4.2 F 4.9

F 1.5 F F F F F F

1.5 3.7 1.6 2.5 2.1 5.6

CFU-GEMM 0 2.4 0.6 0.2 1.8 0 1.0 1.3 0 0 0.4 0 0.6 3.4 0 0.6 5.4 3.0

F 2.1

F 0.5

F 1.3 F 1.9

Abbreviation: N.C., negative control mouse without injected cells. Cord blood CD34+ cells cultured under survival conditions were transduced with Ad5F35-GFP at an m.o.i. of 100, sorted into CD34+GFP+ and CD34+GFP fractions, and transplanted into NOD/SCID mice as described under Materials and Methods. Mouse bone marrow was harvested at 6 (experiment 1) or 5 (experiment 2) weeks posttransplantation and screened for the expression of CD45, CD19, and CD15/33 by flow cytometry. Bone marrow cells were plated in methylcellulose medium with cytokines specifically supporting the growth of human primitive hematopoietic progenitor cells as described under Materials and Methods. Numbers of colonies were counted 14 days later according to standard criteria. Data shown are the averages and standard deviations of colony numbers per dish of 5 dishes.

duction at an m.o.i. of 100. Under these conditions, about 40% of the CD34+ cells were expressing GFP at the time of sorting. We transplanted equal numbers of the CD34+GFP+ and CD34+GFP cells. As shown in Table 3, the CD34+GFP+ and CD34+GFP cells showed no significant difference in reconstitution capacity as measured by the percentages of human CD45+ cells in transplanted NOD/SCID mouse bone marrow (7.8 F 5.2% versus 11.3 F 10.2%, P = 0.36, t test). All mice with human cell reconstitution exhibited both myeloid and lymphoid cell engraftment. To assess whether the Ad5F35-GFP-transduced NOD/SCID repopulating cells were capable of generation of primitive hematopoietic progenitor cells in the NOD/SCID mouse bone marrow, we plated mouse bone marrow cells in methylcellulose medium with cytokines specifically supporting the growth of human progenitor cells. Bone marrow cells from the recipients transplanted with the CD34+GFP+ cells did contain cells with colony-forming unit – granulocyte – macrophage (CFU-GM), burst-forming unit – erythroid (BFU-E), and colony-forming unit – granulocyte, erythrocyte, macrophage, and megakaryocyte (CFUGEMM) capacity. We observed no reduction in the numbers of colony-forming cells compared with the recipients transplanted with the CD34+GFP cells (Table 3). Collectively, our data suggest that NOD/SCID repo-

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pulating cells can be efficiently transduced by the Ad5F35-GFP vector without any loss of functionality.

DISCUSSION Our findings are consistent with previous reports that Ad5-based vectors retargeted to Ad35 tropism or Ad35based vectors allow efficient gene transfer into hematopoietic cells with primitive immunophenotype in a CARindependent manner [7,25,28,29]. Importantly, our data show that the Ad5F35-GFP vector allowed efficient gene transfer into nondividing primitive cells with high levels of CD34 antigen expression, whereas the Ad5-GFP vector preferentially transduced the dividing progenitor cells with lower levels of CD34 expression. Furthermore, primitive hematopoietic cells were efficiently transduced by the Ad5F35-GFP vector without compromising NOD/ SCID reconstitution capacity, while the Ad5-GFP vectortransduced primitive cells contained no or barely detectable levels of NOD/SCID reconstitution capacity. Adenoviral vector-mediated transient gene expression in HSCs is a desirable approach for mechanistic studies and therapeutic manipulation of HSC self-renewal and differentiation. Several groups have investigated the potential of adenoviral vector-mediated gene delivery into primitive hematopoietic cells by using fiber-gene-unmod-


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ified or -modified vectors [7,9 – 18,25,28,29]. As most of the repopulating HSCs are quiescent [2,35 – 41], it is of key importance that adenoviral vector-mediated gene transfer can be demonstrated in quiescent or nondividing primitive hematopoietic cells with repopulating capacity. It has been controversial whether HSCs can be efficiently transduced by adenoviral vectors [7,10,17,18]. The efficiency of adenoviral vector-mediated gene delivery is primarily dependent on the availability of the cellular receptors on host cells. The expression of the CAR was demonstrated in erythroid and myeloid progenitors [34], but primitive hematopoietic cells, in particular the freshly isolated bone marrow CD34+CD38 cells, do not express sufficient levels of CAR [6 – 8,34]. Consistent with a previous report [34], we show that CAR expression can be up-regulated in differentiated progenitor cells but not in cells maintaining primitive immunophenotype during cytokine stimulation in in vitro culture. In agreement with these observations, efficient gene transfer by fibergene-unmodified Ad5 vectors was reported in expanded hematopoietic progenitor cells [14]. To improve Ad5 vector-mediated gene transfer into primitive hematopoietic cells, strategies including prolonged incubation of primitive hematopoietic cells with high m.o.i. of Ad5based vectors, stimulation of primitive cells with cytokines or histone deacetylase inhibitors, and formation of complexes between Ad5 vectors and polycationic reagents have been explored [6,10,12 – 17]. These conditions most likely improved gene transfer into primitive hematopoietic cells in a receptor-independent manner. However, as exemplified in the SuperFect supported transduction protocol, high m.o.i. of the Ad5-based vectors are still required for sufficient gene delivery into the NOD/SCID repopulating cells, and the nondividing CD34+ cells were transduced less efficiently than dividing CD34+ cells [10]. Here we show that following a brief incubation with the Ad5-GFP vector without polycationic reagents, at m.o.i. between 100 and 500, up to 25% of cord blood CD34+ cells were expressing high levels of GFP. The Ad5-GFP-transduced cells were predominantly proliferating cells and exhibited nearly undetectable NOD/SCID mouse repopulating capacity. Our data are supported by the observations that Ad5-based vectors allowed selective gene expression and elimination of contaminating carcinoma cells in bone marrow or mobilized peripheral blood stem cell sources from cancer patients, whereas the stem cell capacity of purged samples was intact [8,42,43]. Recently, Ad5 vectors with fiber retargeted to species B2 adenovirus tropism or species B2 Ad35-based vectors have been demonstrated to have high efficiency of gene transfer into hematopoietic cells with primitive immunophenotype [7,24,25,28,29]. This is consistent with the finding that species B2 adenoviruses utilize ubiquitously expressed CD46 as a cellular receptor [22,23], and we also showed that CD46 was highly expressed in all cord blood


ARTICLE CD34+ cells tested (Fig. 3). However, it has been unclear whether the successfully transduced cells with primitive immunophenotype contain NOD/SCID repopulating cells, as only a small fraction of freshly isolated CD34+ or CD34+CD38 cells exhibit repopulating capacity in the NOD/SCID mice transplantation assay [44]. Furthermore, it has been suggested that transduction with adenoviral vectors, including the fiber-retargeted vectors, may diminish the functionality of the primitive hematopoietic progenitor cells [7,18]. As most of the repopulating HSCs and primitive leukemic cells are nondividing upon isolation [2,35 – 41], gene delivery into nondividing cells is of crucial importance. Our data show that the Ad5-GFP vector preferentially transduced the proliferating CD34+ cells, which is consistent with the report that proliferating cells allow superior binding and uptake of Ad5-based vectors [45]. However, our results appear to contradict a previous report that an Ad5-based vector mediated equal gene delivery efficiency into cultured bulk CD34+ cells and quiescent cells with CD34+CD38 immunophenotype [16]. Several reasons may account for this difference. First, the cell tracking strategy used in our assay assessed the cell proliferation history of each individual cell, whereas the analysis of cellular DNA content and Ki-67 expression used in the other study allows only a snapshot of the cell cycle status. Proliferating cells may reenter the G0 phase of the cell cycle following cell division [37]. Second, a shorter transduction protocol was used in this study compared with the study by Neering et al. [16]. A longer incubation period with high m.o.i. of Ad5-based vector may allow gene transfer into primitive hematopoietic cells in a CAR-independent manner. Third, different regulatory sequences were used for controlling transgene expression, and this may explain different expression levels in various subpopulations of CD34+ cells [11,13]. In contrast to the Ad5-GFP vector, our findings show that dividing CD34+ cells are preferentially transduced by the Ad5F35-GFP at an m.o.i. of 20 (Fig. 4B), whereas nearly equal frequencies of the GFP-expressing cells in dividing and nondividing CD34+ cells can be achieved at m.o.i. between 100 and 500. The abundant expression of CD46 allows efficient uptake of the Ad5F35GFP vector. In addition, the Ad5F35-GFP vector may also exhibit different intracellular trafficking pathways compared with the Ad5-GFP vector such that relatively low gene transfer efficiency into nondividing cells can be overcome at increased m.o.i. However, although all CD34+ cells expressed the CD46 antigen, the percentage of the GFP-expressing cells did not increase proportionally with the increased m.o.i. of the Ad5F35-GFP vector (Figs. 2 and 3), suggesting that a subset of the CD34+ cells was not permissive to successful gene transfer by the Ad5F35-GFP vector. Currently, it is unclear whether this is due to a blockage of vector uptake or intracellular trafficking or to inefficient transcription and/or transla-

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tion of successfully delivered vector genomes in a particular subset of CD34+ cells. Future studies distinguishing between these possibilities are planned. Our results demonstrate that gene transfer into NOD/ SCID repopulating cells can be achieved at relatively low m.o.i. of the Ad5F35-GFP vector. To our knowledge, this is the first report that an Ad5F35-based vector can allow efficient gene delivery into NOD/SCID repopulating cells. The functional outcome of gene delivery studies into primitive hematopoietic cells is likely dependent on the cell culture conditions and vector design. As primitive hematopoietic cells differentiate under most in vitro culture conditions, the stem cell capacity can be lost or severely diminished during adenoviral vector gene transfer protocols. Our study was performed under serum-free conditions with the support of MGDF alone, which supports the survival and minimal proliferation of primitive progenitor cells, and the primitive cells can be successfully transduced and sorted within 2 days of culture. Importantly, large numbers of successfully transduced cells containing a substantial fraction of nondividing cells were sorted following transduction with the Ad5F35GFP vector at m.o.i. between 100 and 200, and the GFP+ cells showed similar reconstitution levels in NOD/SCID mice compared with the GFP cells and mock-transduced cells. In contrast to previous reports [17,18,33], no cytopathic effect was observed in our study. Thus, the stem cell capacity was likely better preserved compared with studies in which CD34+ cells were cultured in serumcontaining medium with multiple cytokine stimulation [6,7,12,13,15 – 17,25]. Alternatively, the design of the transgene expression cassette can affect the levels of transgene expression between different subsets of primitive hematopoietic cells [11,13]. Previous studies utilizing an Ad5F35-based vector showed that successful gene delivery is not necessarily translated into gene expression in CD34+ cells [7]. Therefore, the functional outcome of the transgene-expressing CD34+ cells following adenoviral vector gene transfer may differ, depending on the regulatory sequences controlling transgene expression. In conclusion, our findings suggest that Ad5F35-based vectors will enable transient genetic manipulation of NOD/SCID repopulating cells. By using this approach, transient expression of cytokine receptors, cytokine-dependent kinases, and transcription factors can be applied to promote HSC expansion or to direct the HSC differentiation along a specific lineage.

MATERIALS AND METHODS Adenoviral vectors and antibodies. The adenoviral vectors used in this study are depicted in Fig. 1. Both vectors encode a GFP expression cassette in the E1 region. The Ad5F35-GFP vector encoding the GFP gene under the upstream control of the murine Pgk1 gene promoter and downstream control of the rabbit h-globin gene IVS 2 and polyadenylation signal was generated using the fiber-gene-modified AdEasy system. The modification

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of the AdEasy system and the generation of the Ad5F35-GFP vector will be described elsewhere [27]. This vector harbors the engineered fiber gene encoding the Ad5 fiber tail domain and Ad35 fiber shaft and knob domains and therefore infects target cells in a CAR-independent manner. The Ad5-GFP vector carries an unmodified Ad5 fiber gene and therefore requires CAR and av integrins for efficient cellular infection [9]. In the Ad5-GFP vector, the GFP gene expression cassette is under the upstream control of the murine Pgk1 gene promoter and downstream control of the human h-globin gene IVS 2 and polyadenylation signal. Both vectors were expanded in 293 cells, purified by two successive CsCl centrifugations, and stored at 70jC until use. Functional titration of the viral stocks was performed by limiting dilution assays on 293 cells, and physical particles were quantified according to the method described by Mitterander et al. [46]. The functional titers were between 1 1010 and 1 1011 infectious units (IU)/ml for the Ad5-GFP and the Ad5F35-GFP vector preparations. The ratio between physical particles and functional IU was about 10:1 in all stocks used. The anti CAR mAb (clone RcmB) was purchased from Upstate Biotechnology (Charlottesville, VA, USA) and the rat IgG blocking antibody was purchased from the Jackson Immunoresearch Laboratories (West Grove, PA, USA); all other mAbs used in this study were purchased from Becton – Dickinson Immunocytometry Systems (Mountain View, CA, USA). Culture and transduction of cord blood CD34+ cells. The umbilical cord blood CD34+ cells were isolated and cultured under serum-free conditions as described previously [10]. In brief, CD34+ cells were isolated from pooled cord blood samples collected after normal delivery by using a CD34+ progenitor cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity was routinely above 95% as assessed by flow cytometric analysis. CD34+ cells were cultured in serum-free X-vivo 15 (BioWhittaker, Walkersville, MD, USA) containing 1% BSA (Stem Cell Technologies, Vancouver, Canada), 2 mM L-glutamine, 100 AM 2-mercaptoethanol, 100 units/ml penicillin, and 100 Ag/ml streptomycin in 48well plates at a cell density of 1 105 cells in 300 Al medium per well. In addition, cells were supported either with an early acting cytokine cocktail consisting of MGDF, SCF, and Flt3 ligand (proliferating condition) or with MGDF alone (survival condition). The MGDF and Flt3 ligand were used at 50 ng/ml and SCF at 100 ng/ml. After overnight culture, adenoviral vectors corresponding to m.o.i. of 20, 100, or 500 were added and mixed well. Three hours later, unbound viruses were washed away and cells were resuspended in 500 Al of cytokine-containing fresh medium. The GFP expression was assessed 48 h posttransduction in flow cytometry by staining cells with fluorochrome-conjugated anti-CD34 mAb or isotype-matching control antibodies in the presence of 7-aminoactinomycin D (7-AAD) at 1 Ag/ml. Living cells negatively stained for 7AAD were gated for CD34 and assessed for GFP expression analysis. The duration of GFP expression in the CD34+ cells cultured under proliferating conditions was assessed every second day as described above. Analysis of CAR and CD46 expression. On day 1 and day 3 of the culture (at the time of viral infection or GFP expression analysis), the expression of CAR and CD46 was measured on mock-transduced cells in combination with CD34 antigen staining. Prior to CAR staining, cultured cells were blocked by rat IgG antibody, followed by staining with RcmB mAb and PE-conjugated rat anti-mouse IgG1 mAb. Subsequently, cells were stained with APC-conjugated anti-CD34 mAb. Control staining for CAR was done by omitting the RcmB mAb. HeLa cells were used as positive control for RcmB staining. The CD46 staining was performed with FITCconjugated anti-CD46 mAb together with APC-conjugated anti-CD34 mAb as described above. CAR expression was compared between cells with 50% lowest CD34+ expression and cells with 50% highest CD34+ expression (Fig. 3). Cell division tracking experiments. Prior to the initiation of cell culture, cord blood CD34+ cells were stained using a PKH26 red fluorescence cell linker kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. A portion of the stained cells was fixed at 4jC in phosphate-


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buffered saline containing 2% formaldehyde and 0.2% glutaraldehyde as a control for nondividing cells. PKH26-stained cells were cultured and transduced with the Ad5-GFP and the Ad5F35-GFP vectors as described above. Two days after transduction, cells were stained with APC-conjugated anti-CD34 mAb and analyzed for GFP expression in dividing and nondividing CD34+ cells. The nondividing cells were defined as the cells with PKH26 intensity higher than the peak channel of the fixed cells, and the dividing cells were defined as cells with the PKH26 intensity lower than the peak channel of cells cultured under proliferating conditions (Fig. 4A). NOD/SCID reconstitution assay. The NOD/SCID mice, which originated from The Jackson Laboratory (Bar Harbor, ME, USA), were housed in sterile microisolator cages in ventilated racks and supplied with sterile food, acidified water, and bedding. Eight- to 11-week-old animals were irradiated with a sublethal dose of 350 rad within 24 h prior to transplantation. Transplanted mice were provided with the antibiotic ciprofloxacin at 100 Ag/ml in drinking water. Cord blood CD34+ cells were cultured under survival conditions and transduced with the Ad5-GFP and Ad5F35-GFP vector as described above; cell sorting was performed at 40 h posttransduction with or without CD34 staining. For experiments presented in Fig. 5, GFP+ cells were sorted and animals were transplanted with 30,000 to 50,000 sorted GFP+ cells together with 1 106 irradiated (1500 rad) cord blood mononuclear cells as accessory cells. The purity of sorted cells was >99.5% as assessed by reanalysis in all experiments. For experiments presented in Table 3, cells were sorted into GFP+CD34+ and GFP CD34+ cells following the Ad5F35-GFP transduction. Each mouse was transplanted with 1.0 105 (Expt 1) or 2.0 105 (Expt 2) cells via tail vein injection. Recipient mice were killed at 5 to 6 weeks after transplantation by CO2 inhalation, and bone marrow from the femurs was harvested. The human cell reconstitution in the mouse bone marrow was analyzed according to previously described procedures [47]. In brief, bone marrow cells after lysis of red blood cells with NH4Cl were analyzed for human cell chimerism by staining with APC-conjugated anti-human CD45 mAb, as shown in Fig. 5B. Myeloid reconstitution was measured by costaining with PE-conjugated anti-CD15 and anti-CD33 mAbs, and lymphoid reconstitution was measured by costaining with PE-conjugated anti-CD19 mAb. Costaining with PE-conjugated anti-CD34 mAb assessed the generation of primitive CD34+ cells. For each staining, isotypematched control mAbs were used to set up the background staining. For assessing the human primitive progenitor cells with colony-forming capacity in the NOD/SCID mouse bone marrow, 50,000 or 100,000 bone marrow cells from each mouse were plated in 1 ml of methylcellulose medium (MethoCult H4230; Stem Cell Technologies) supplemented with human SCF (25 ng/ml), granulocyte – megakaryocyte stimulating factor (50 ng/ml), interleukin-3 (25 ng/ml), and erythropoietin (5 units/ml). These conditions specifically support the growth of human progenitor cells. The BFU-E, CFU-GM, and CFU-GEMM were counted after 14 days of culture. Bone marrow cells from nontransplanted NOD/SCID mice were included as negative controls to ensure the human origin of the CFU-Cs.

ACKNOWLEDGMENTS We thank Lilian Wittman, Eva Gynnstam for expert assistance with animal experiments, Anna Fossum, and Dr. Zhi Ma for cell sorting. We are indebted to Dr. Saemundur Gudmundsson and the staff of the Department of Obstetrics and Gynecology, Malmo¨, for collecting umbilical cord blood samples. We thank Drs. Ian K. McNiece, Janet Nichol (Amgen, Thousand Oaks, CA, USA) for generously supplying MGDF and KL, and Dr. Stewart Lyman (Immunex, Seattle, WA, USA) for providing FL for these studies. These studies were supported by grants from the Swedish Cancer Society, the Royal Physiographic Society in Lund, the Crafoord Foundation, the Gunnar Nilsson Cancer Foundation, the Erik PhilipSo¨rensen 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. RECEIVED FOR PUBLICATION NOVEMBER 1, 2003; ACCEPTED DECEMBER 16, 2003.


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