Genetically Modified CD34+ Cells As Cellular Vehicles for Gene Delivery

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Gene Therapy (2000) 7, 43–52  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt CELL-BASED THERAPY RESEARCH ARTICLE Genetically modiﬁed CD34+ cells as cellular vehicles for gene delivery into areas of angiogenesis in a rhesus model

JGo´mez-Navarro1, JL Contreras2, W Arafat1, XL Jiang2, D Krisky3, T Oligino3, P Marconi3, B Hubbard2, JC Glorioso3, DT Curiel1 and JM Thomas2 1Gene Therapy Center, Birmingham; 2Transplant Immunobiology, Department of Surgery, Birmingham, AL; and 3Department of Molecular Genetics and Biochemistry, E1240 Biomedical Science Tower, Pittsburgh, PA 15261, USA

To develop a cellular vehicle able to reach systemically dis- lets impregnated with vascular growth factors underwent seminated areas of angiogenesis, we sought to exploit the necrosis or accelerated regression after administration of natural tropism of circulating endothelial progenitor cells ganciclovir. Importantly, the whole intervention was perfectly (EPCs). Primate CD34+ EPCs were genetically modiﬁed with well tolerated. The accessibility, easy manipulation, lack of high efﬁciency and minimal toxicity using a non-replicative immunogenicity of the autologous CD34+ cell vehicles, and herpes virus vector. These EPCs localized in a skin autograft tropism for areas of angiogenesis render autologous CD34+ model of angiogenesis in rhesus monkeys, and sustained circulating endothelial progenitors as ideal candidates for the expression of a reporter gene for several weeks while exploration of their use as cellular vehicles when systemic circulating in the blood. In animals infused with autologous gene delivery to those areas is required. Gene Therapy CD34+ EPCs transduced with a thymidine kinase-encoding (2000) 7, 43–52. herpes virus, skin autografts and subcutaneous Matrigel pel-

Keywords: rhesus monkey; CD34 cell; gene transfer; herpes simplex virus; herpes simplex virus thymidine kinase; anti-angiogenesis

Introduction ation of vectors have limited the overall efficacy of gene therapy approaches. A variety of strategies has been developed to accomplish As an alternative, cells have also been employed as gene therapy for cancer and other systemic diseases and vectors. In this approach, therapeutic genes are trans- conditions characterized by the development of angio- ferred to isolated cells ex vivo and the transduced cells genesis, including inflammatory diseases and organ are reimplanted in vivo. In this manner, the genetically 1–4 engrafting. Fundamental to the clinical implementation modified cell becomes itself the ultimate vector for gene of these strategies is the development of vector systems delivery.12–14 A variety of genes can conceivably be intro- that allow direct in vivo gene transfer to disseminated loci duced in candidate cell vehicles, and expression of these of target tissues. To this end, both viral and non-viral vec- gene payloads can be obtained in the ultimate environ- 5–8 tors have been employed. In general, despite their util- ment where these cells finally localize. A loco-regional ity in selected contexts, currently available viral vectors effect subsequent to the expression of the therapeutic and liposomes have been quite limited in their ability to gene can thus be achieved in areas otherwise hardly accomplish highly efficient gene delivery to relevant tar- accessible to gene transfer. For example, we have pre- get cells in vivo. Most importantly, the lack of vectors viously shown that human umbilical vein endothelial with the capacity to achieve cell-specific targeting has cells (HUVECs) genetically modified with an adenovirus broadly restricted the implementation of gene therapy in encoding HSV thymidine kinase (tk) may be used in vivo many disease contexts, and notably in disseminated as cellular vehicles for the induction of a cytotoxic malignant tumors. The innate and adaptive immune bystander effect on tumor cells.15 responses against viral vectors and accompanying Cellular vehicles offer numerous advantages as vectors inflammation have also presented formidable barriers, and have had therefore historical precedence. However, even in the neoplastic disease context, for significant gene stringent criteria, largely unmet, are required for delivery and expression, and thus for successful clinical developing a clinically feasible cellular vehicle vector sys- 5,9–11 translation. Thus, shortcomings in the current gener- tem: (1) simple and non-invasive harvest from the patient; (2) non-deleterious manipulation and/or expansion in culture; (3) high levels of cell transduction with Correspondence: JM Thomas, Transplant Immunobiology, Department of Surgery, 1808 Seventh Avenue South, BDB 563, Birmingham, AL available vectors; (4) low risk and easy reimplantation; 35294, USA and (5) adequate cellular tropism. In the context of a Received 19 July 1999; accepted 9 August 1999 paucity of candidate cells that could meet the stringent Endothelial progenitors for systemic gene delivery J Gomez-Navarro et al 44 requirements mentioned above for a clinically useful less than 1% of cells transduced with the control T3 vec- cellular vehicle, it has recently been reported that the tor, non-expressing the reporter gene, were positive in CD34+ leukocyte fraction of human peripheral blood con- this analysis. Similarly high efficiency was obtained with tains putative endothelial progenitors which originate in CD34+ cells isolated from bone marrow of a non-human the bone marrow and localize in areas of post-ischemic primate (data not shown). Thus, an exceptionally high angiogenesis.16 Additional evidence from a canine model efficiency of transduction can be achieved in primate confirmed that a subset of donor bone marrow cells can CD34+ cells with a non-replicative herpes virus after a be mobilized to the peripheral circulation and colonize short incubation in serum free and stromal cell free endothelial flow surfaces.17 In both reports, purified conditions. CD34+ cells were shown to differentiate in vitro into mature endothelium.16 In addition to these studies based Transduction of CD34+ cells with replication-deficient on animal models of transplantation of CD34+ cells, a var- herpes virus has low toxicity in vitro iety of other experimental observations support the inti- To determine the toxicity of multiple-gene deleted HSV mate role of CD34+ cells in angiogenesis, including stud- vectors in CD34+ cells, we continuously exposed human ies on implanted vascular grafts18–24 and molecularly CD34+ cells in vitro to the TOZ.1 herpes virus vector for based histological25 and embryological studies.26–36 up to 7 days, and measured viable cells at 2, 4 and 7 days Several threads of evidence converge therefore to using a quantitative colorimetric assay. There was no sig- strongly support the existence in the adult of circulating nificant decrease in the number of cells except at day 2 progenitor cells with endothelial potential.17 at an MOI of 30, a dose of virus one order of magnitude Transduction of human and non-human primate higher than needed for an optimal level of gene transfer CD34+ cells, though, has been hampered by its consist- (Figure 2). On the contrary, a virus-related cell prolifer- ently low efficiency.37–44 Unfortunately, maneuvers for ation was observed mostly at day 2. Analogous experi- improving transduction in vitro, such as prolonging ments in Vero cells showed a similar lack of deleterious exposure to viral vectors, stimulating cell proliferation, effects on cell survival (data not shown). Cytotoxicity or culturing on stromal cells, affect the phenotypic fea- studies revealed therefore minimal or null deleterious tures of progenitor cells in undesirable ways.45,46 To effect of TOZ.1 on cell survival at the optimal MOI of 3 explore the concept of systemic gene delivery into areas or less. of angiogenesis, we sought to use cellular vehicles nat- urally endowed with angiogiogenic tropism. Fundamen- Autologous transplanted CD34+ cells home into areas of tal to this goal, we have shown that recombinant, non- angiogenesis in an in vivo model replicative herpes virus vectors (HSV) provide a novel To determine the capacity of genetically modified CD34+ means to modify with high efficiency and low toxicity cells to localize into areas of angiogenesis, we transduced human and nonhuman primate CD34+ cells genetically. them ex vivo with the highly efficient HSV-based vector The remarkable transducing ability of the vector facili- TOZ.1, which encodes the reporting gene lacZ. The modi- tates short-term incubations for genetic manipulation of fied CD34+ cells were autologously transplanted in three CD34+ angioblasts, avoiding undesired cellular pheno- rhesus macaques bearing 2-day-old skin autografts. typic changes in culture. Furthermore, we have observed, Approximately 1–2 × 106 CD34 cells/kg were infused in by infusing genetically modified CD34+ cells in rhesus each animal. Considering the purity of the cell prep- macaques bearing skin autografts, the expression of aration (70–85%), the efficiency of transduction (90–95%), reporter genes and the presence of vector genome in and the estimated blood volume (65 ml/kg), the esti- areas of angiogenesis. Importantly, the accessibility, easy mated concentration of transduced CD34+ cells in these manipulation, lack of immunogenicity of the autologous 3–4 kg animals was between 9600 and 11000 cells/ml. CD34+ cell vehicles, and tropism for areas of angiogenesis This is in contrast to an estimated concentration in non- of such a cell render autologous CD34+ endothelial mobilized animals of 50 CD34+ cells/ml (assuming a per- progenitors ideal candidates for use as cellular vehicles centage of 0.5% of CD34+ cells and 10000 mononuclear targeted against areas of angiogenesis distributed cells/ml). Additionally, as an alternative model of angio- systemically. genesis, subcutaneous pellets of either Matrigel or Gel- foam were impregnated with the potent angiogenic growth factors VEGF and bFGF. Results Biopsies of both skin grafts and pellets, obtained at several time points, were stained with X-gal to reveal activity Human CD34+ cells are transduced with high efficiency of the lacZ gene-encoded beta-galactosidase. Small, by a replication-deficient herpes virus round, intensely blue cells were identified in neovascular To explore novel means of efficiently transducing human structures (Figure 3a, b and c), both in grafts and pellets. and non-human primate CD34+ cells, we evaluated a Staining with anti-factor VIII antibodies confirmed the second-generation herpes virus vector. Its main features vascular character of these tube-like structures. Thus, the are its being non-replicative by virtue of several deletions possibility of circulating CD34+ cells to home into areas in genes essential for replication and its reduced cytotox- of angiogenesis was confirmed in this model. icity obtained by additional early gene deletions (Figure A key aspect of our hypothesis is the localization of 1a). After an exposure for 12 h of CD34+ cells obtained endothelial progenitors in areas of angiogenesis. In situ from a mobilized normal human donor to the TOZ.1 her- hybridization was performed as a confirmatory technique pes vector at 0.3 and 3 p.f.u. per cell, 11.7% and 99.9% of to obtain evidence for the presence of the genetically cells expressed the reporter gene lacZ, respectively, modified endothelial progenitors in the target skin auto- according to a fluorescence-activated cell sorter (FACS) grafts. In situ hybridization with a probe specific for a analysis using FDG (Figure 1b, population 4). In contrast, HSV vector sequence demonstrated nuclear staining of

Gene Therapy Endothelial progenitors for systemic gene delivery J Gomez-Navarro et al 45

Figure 1 Genetic modification of endothelial progenitors by herpes virus vectors (a) For constructing recombinant herpes virus vectors, a typical HSV- 1 150000 kb viral genome has been deleted in genes essential for replication (ICP4 and ICP27) and genes additionally related to viral cytotoxicity (ICP22, UL41), rendering them substantially less toxic and allowing longer transgene expression. Further, the reporter gene lacZ and the protoxin gene thymidine kinase were inserted under the control of HSV immediate – early gene promoters ICPO and ICP4, respectively. The structure of TOZ.1 is − − − − − − − ICP4 , ICP22 , ICP27 ,UL24 : ICP4TK, UL41 : ICP0 lacZ. T3 was used as negative control for lacZ staining, and its structure is ICP4 , ICP22 , − − ICP27 ,UL24 : ICP4TK The diagram shows the vectors backbone, deleted genes, and transgenes. Methods for constructing HSV-derived vectors have been described elsewhere.60 Open squared boxes represent HSV sequence deletions. Black boxes represent expression cassettes. (b) Expression of lacZ as determined by production of the fluorescent metabolite of FDG following infection by TOZ.1. Frozen human CD34+ cells were cultured for 1 h and incubated for 12 h (1) in serum-free media alone; (2) in the presence of the non-lacZ expressing herpesvirus T3; and either (3) with 0.3 p.f.u. or with (4) 3 p.f.u. of the lacZ expressing herpes virus TOZ.1. Next, they were stained with FDG for flow cytometry analysis.

graft cells (Figure 3f). In situ hybridization on HSV- Sustained low levels of transgene expression in infected CD34+ cells with an irrelevant probe (specific for peripheral blood and bone marrow plasmid DNA) was negative (Figure 3d), as was in situ The use of genetically modified endothelial progenitors hybridization with the lacZ gene probe on grafts from in vivo requires that expression of encoded transgenes be animals which did not receive genetically modified long enough to assure their effect. Therefore, we sought CD34+ cells (data not shown). Evidence for the presence to detect transgene expression in blood and bone marrow of the HSV vector and for the expression of the lacZ in animals receiving infusions of HSV-infected CD34+ reporter gene can thus be observed in skin autografts, cells. To examine the duration of expression of the while angiogenesis was taking place therein. reporter gene lacZ, the ex vivo-modified autologous mon-

Gene Therapy Endothelial progenitors for systemic gene delivery J Gomez-Navarro et al 46 In vivo administration of CD34+ cells modified by a replication-deficient herpes virus appears to be safe During the first 12 months after infusion of CD34+ cells, no symptom or sign of toxicity has been observed in any of the three different primates treated with TOZ.1-transduced cells. Specifically, there was no change in behavior, weight or vital signs. Furthermore, there was no alter- ation in any standard hematocytometric or biochemical study, including liver and renal function biochemical profiles, with the exception of a reversible and minor increase in alkaline phosphatase during ganciclovir treatment (data not shown). However, to fully prove the safety of the replication-deficient herpes virus vector further studies are necessary.

Discussion We have examined, in a rhesus primate model, the possibility of genetically modifying CD34+ endothelial progenitor cells and have used them as cellular vehicles for Figure 2 HSV exhibits minimal cytotoxicity on human CD34+ cells. To systemic delivery into areas of angiogenesis. We found evaluate the cytotoxicity of the TOZ.1 vector, a cell survival assay was + + that human and non-human primate CD34 cells can performed. Frozen human CD34 cells were cultured for 1 h and then indeed be transduced with high efficiency and minimal infected with the herpes virus TOZ.1 at MOIs of 0, 0.3, 3 and 30. Next, toxicity in vitro with a novel, non-replicative herpes virus cells were plated in a 96-well plate, in quadruplicate wells, in media con- vector. Furthermore, we have observed, by infusing gen- taining the growth factors IL-3, IL-6 and SCF. Cell survival was deter- + mined after 2, 4 and 7 days of continuous exposure in culture by means etically modified CD34 cells in rhesus macaques bearing of a colorimetric cell proliferation assay. Two independent experiments skin autografts, the expression of reporter and toxin were performed. Bars, mean percentage of surviving cells in a representa- genes and the presence of vector genome in areas of angi- ± tive experiment, s.d. of the mean. There was no significant decrease in ogenesis. Importantly, the autologous transplantation of the number of cells except at day 2 at an MOI of 30, a dose of virus one transduced CD34+ cells is well tolerated by the monkeys, order of magnitude higher than needed for an optimal level of gene transfer. On the contrary, a virus-related cell proliferation was observed mostly and the expression of the reporter gene is sustained for at day 2. Analogous experiments in Vero cells showed a similar lack of several weeks. We have not yet analyzed or optimized, deleterious effects on cell survival (data not shown). Cytotoxicity studies however, the quantitative relation between the number of revealed therefore minimal or null deleterious effect of TOZ.1 on cell sur- infused cells, the quantity of transgene, and its selective vival at the optimal MOI of 3 or less. expression in target areas of angiogenesis. The question of whether HSV-transduced CD34+ cells home specifically to sites of angiogenesis requires further resolution. This important practical issue is the subject of current key cells were introduced intravenously and the animals studies in a tumor mouse model, more amenable to this were monitored by flow cytometry of peripheral blood quantitative and invasive study involving numerous ani- MNC to detect the proportion of FDG-positive cells in mals. This uncertainty however does not alter the essence the circulating CD34+ population. We were able to detect of the observations delineated herein, particularly the gene in both peripheral blood and bone marrow aspi- since gene expression in CD34+ cells can be regulated by rates obtained serially for a period of weeks (3 weeks in endothelial cell promoters. two animals, 6 weeks in an additional animal) (Figure 4). Efficient transduction of normal and malignant human In contrast, analysis of an identically gated cell popu- hematopoietic cells, including CD34+ cells, has been pre- lation in mobilized but non-transplanted animals did not viously reported.47 Levels of 70–100% cell transduction reveal any FDG+ cells. were achieved with a disabled infectious herpes virus that is restricted to a single cycle of replication (DISC- Skin autografts in macaques infused with endothelial HSV). Although toxicity appeared to be low, the impact progenitors genetically modified with a tk-encoding HSV of viral replication in hematopoietic cells and the effects are rejected after treatment with ganciclovir of non-attenuated expression of viral genes have not been As an additional way to verify the expression of genes completely characterized. TOZ.1, the herpes virus vector contained in the transplanted CD34+ cells in the target we used, is non-replicative by virtue of the deletion of angiogenesis areas, and to determine if we could exploit two genes essential for replication, and has been deleted such expression with therapeutic intent, we infected in two other genes additionally responsible for herpes- CD34+ cells with the TOZ.1 vector, which encodes the related cytotoxicity. As expected, our studies confirmed gene thymidine kinase. Treatment with ganciclovir in a remarkable absence of cytotoxicity of TOZ.1 in CD34+ animals that received the TOZ.1 transduced CD34+ cells and Vero cells in vitro. Thus, recombinant herpes virus induced the necrosis of skin autografts (Figure 5a) and TOZ. 1 offers some theoretical safety advantages with the disappearance of the subcutaneous pellets (Figure respect to other non-deleted herpes virus vectors. Recom- 5b). In contrast, in animals treated with ganciclovir but binant adenovirus vectors have also been used for transplanted with non-transduced cells, skin autografts obtaining transient gene expression in CD34+ cells. were unaffected and pellets were evident for longer Efficiency, however, has been generally much lower48 periods of time. than with herpes virus vectors,49–54 with a maximum,

Gene Therapy Endothelial progenitors for systemic gene delivery J Gomez-Navarro et al 47 a b c

d e f

Figure 3 Localization of genetically modified cells in areas of angiogenesis. Photomicrographs of skin autograft biopsies obtained 5 days after autologous transplantation of CD34+ cells transduced with the lacZ-encoding herpes virus TOZ.1. Sections were stained with X-gal and eosin (a and b). Staining with anti-factor VIII antibody confirms the vascular character of the displayed structure (c). In situ hybridization was performed as a confirmatory technique to obtain evidence for the presence of the genetically modified endothelial progenitors in the target skin autografts. Hybridization with a probe specific for a HSV sequence demonstrated nuclear staining of graft cells (f). In the inset, two cells positive for HSV genome are shown. Both seem to be located in the wall of capillary tubes (f). As controls, we performed in situ hybridization on slides containing HSV-infected CD34+ cells using an irrelevant probe (specific for plasmid DNA) (d), or the lacZ gene probe (e). Hybridization with the lacZ gene probe on grafts from animals, which did not receive genetically modified CD34+ cells, was also negative (not shown). somewhat exceptional compared with most other pub- chromosomal integration of viral DNA. In non-dividing lished attempts, of 45% modified CD34+ cells from the cells, however, transgene expression has been observed total number of cells infected.49 Retroviral vectors, while to persist from days (neurons) up to several weeks offering the prospect for long-term expression, are not (muscle).57 Using flow cytometry, we could observe only inefficient in transduction of human CD34+ cells, but expression of the reporter gene lacZ in (non-cycling) they are further restricted to cycling cells for their inte- mononuclear cells of the peripheral blood of transplanted gration.55,56 Unfortunately, conditions found to be animals for at least 3 weeks. This analysis, which was less required for improving retrovirus gene transfer, such as invasive than sampling the autograft or subcutaneous stimulation with growth factors for inducing active cell pellets, was complemented by the indirect observation of cycling and prolonged incubation with viral vectors, may HSV-thymidine kinase expression at the target angiogen- critically alter the phenotype and function of target cells. esis areas, as suggested by disappearance or necrosis of In fact, differentiation and loss of desired properties of pellets or skin autograft after ganciclovir treatment. Of stem cells occurs frequently, consequently minimizing note, the prodrug treatment was begun 2 weeks after the relevance of the discreet improvements obtainable in transplantation of transduced CD34+ cells. Thus, herpes gene transfer.46 Thus, recombinant, replication-deficient virus can provide sufficiently sustained expression of herpes virus offers advantages over currently available genes of interest in CD34+ cellular vehicles for appli- alternative vectors, which have limitations for achieving cations that require relatively short-term interventions. It efficient gene transfer of CD34+ cells in a way that main- should be noted that we have not yet directly examined tains the phenotypic and functional integrity of freshly the immunogenicity of HSV-transduced CD34+ cells by isolated CD34+ cells. cytotoxic T lymphocyte analysis. Repeated adminis- The duration of expression of genes encoded by herpes tration of CD34+ cells is a practical possibility, the virus recombinant vectors in dividing cells should be lim- implementation of which will require studying the ited, given that the virus life cycle does not include immune response associated with transplantation of

Gene Therapy Endothelial progenitors for systemic gene delivery J Gomez-Navarro et al 48

Figure 4 Sustained low levels of transgene expression in peripheral blood and bone marrow CD34+ cells from rhesus monkeys were transduced with the lacZ-encoding herpes virus TOZ.1 and autologously transplanted. Mononuclear cells from the peripheral blood were obtained at different time points. Cells were stained with an anti-CD34 antibody and exposed to the galactosidase reagent FDG, and double positives were measured by ﬂow cytometry. We were able to detect gene expression (as measured with the ﬂuorescent substrate of FDG) in both peripheral blood and bone marrow biopsies obtained serially for 3 weeks in two animals, and 6 weeks in an additional animal. A representative curve is shown.

genetically modified autologous cells. Interestingly in this regard, CD34+ cells have been associated with induction of immune tolerance to highly immunogenic alloantigens.58 At this point, further studies are certainly needed to define the broad biodistribution of endothelial progenitors after autologous CD34+ cell transplantation. Ulti- mately, the biodistribution of infused cellular vehicles will determine the therapeutic index of delivering genes with cytotoxic or anti-vascular effect by using our proposed approach. In this regard, the expression of a toxin gene in the endothelial progenitors infused in the skin graft-bearing monkeys was remarkably well tolerated but, at the same time, efficacious enough for inducing graft necrosis. Of note, the parallel distribution of lacZ positive cells between blood and bone marrow in our Figure 5 Effects of the expression of a toxic gene by CD34+ endothelial studies is probably biased by the fact that it was difficult progenitors. Rhesus monkeys were autologously transplanted with CD34+ to obtain bone marrow biopsies of enough volume with- cells transduced with the thymidine kinase-encoding herpes virus TOZ.1. After 15 days, treatment with ganciclovir, 10 mg/kg/day for 14 days, was out the inclusion of contaminating blood, simply from + administered. One animal did not receive CD34 cells and served as con- the trauma of the procedure. Nonetheless, the observed trol. (a) A shortening of the period during which pellets were palpable lack of toxicity confirms that the selectivity of tropism was observed in the transplanted group. (b) Sequence of changes that achievable by the endothelial progenitors, yet to be occurred during the treatment. The autograft was successfully accepted determined, does not compromise the feasibility of our and vascularized by 4–5 days after transplant. Subsequently, the treat- gene delivery approach. Furthermore, it is conceivable ment with ganciclovir at 14–28 days after transplant induced a progress- that the therapeutic genes used for modifying the endo- ive necrotic process that was complete 5 days after the treatment ended. thelial progenitors can be put under control of regulatory sequences specifically activated in proliferating endo- hematopoietic progenitors and differentiated mature thelium, thus limiting the expression of the therapeutic cells. Although destiny of the progeny of transduced genes only to those endothelial progenitors that have CD34+ cells may be relevant in certain contexts, however, localized into areas of active angiogenesis. we planned ultimately to kill transduced cells by treat- Three transplanted macaques tolerated the entire pro- ment with ganciclovir, and therefore did not studied this cedure without complications, and more than 18 months issue further at this point. later do not present any evidence of hematological, Whereas a large number of attempts have sought to immunological, or solid organ toxicity. With regard to overcome the limitations of currently available viral and the function of the genetically modified CD34+ cells, we non-viral vectors, particularly using ex vivo gene transfer have not yet performed in vitro or in vivo assays to deter- into parenchymal cells, little work has been proposed to mine their capacity to proliferate and differentiate into employ ‘cellular vehicles’ in an autologous context.

Gene Therapy Endothelial progenitors for systemic gene delivery J Gomez-Navarro et al 49 Further, although the homing capacity of CD34+ endo- described. Of note, purification of viral stocks was done thelial progenitors has been recently described in adult by centrifugation in OptiPrep (Gibco, Gaithersburg, animals,16,17 there is no previous experience with cellular MD, USA). vectors that display the natural ability both freely to cir- culate systemically and localize into areas of angiogensis. Human and primate normal CD34+ cells Our experiments involved skin autografts merely as We sought to develop a vector system that could have models of angiogenesis that could be readily performed potential for rapid translation to clinical use. To avoid in in non-human primates. We believe that the physiology advance the aforementioned problems with interspecies involved, however, has allowed establishment of proof- variation in transduction efficiency, human peripheral of-concept. Our results suggest that CD34+ cells can blood mononuclear cells (MNC) were obtained by leu- indeed localize into areas of angiogenesis and effect the kapheresis of a normal donor mobilized by treatment expression of an incorporated transgene, and allow the with 15 ␮g/kg/day of G-CSF for 5 consecutive days. evaluation of their potential as cellular vehicles with Human CD34+ cells were purified by immunoadsorption capacity for recruitment into areas of angiogenesis. The to 95% purity (Systemix, Palo Alto, CA, USA). Cells were timing of destruction of the autografts and subcutaneous then frozen at −70°C until further use. pellets suggested a local toxic effect induced by the pro- Alternatively, we used non-human primate cells drug ganciclovir that was efficacious even despite a prob- obtained in this case from aspirates of normal bone mar- ably low number of genetically modified CD34+ cells row of similarly mobilized Macacca mulatta. These target implanted therein. This destruction likely occurred by cells were chosen having in mind the rhesus monkey as a transformation of the prodrug into toxic metabolites in relevant animal species to be used in eventual preclinical cells expressing thymidine kinase, with subsequent cell experiments. MNC were isolated by density centrifug- death. This mechanism would be consistent with the local ation over Ficoll–Paque (Pharmacia, Piscataway, NJ, replication of CD34+-derived endothelial cells.16 In USA) and CD34+ cells were isolated on a Ceprate (CellPro addition, toxic bystander effects from ganciclovir metab- Inc, Bothell, WA, USA) immunoaffinity column accord- olites, ischemia from vascular damage and occlusion, and ing to the manufacturer’s protocol. inflammatory reactions could all contribute to the necrosis observed. Beyond the dependence on ganciclovir Transduction of CD34+ cells treatment, we have not yet precisely discriminated a pri- Highly enriched CD34+ cells were plated in 1 ml of mary mechanism underlying the necrosis. Support for a serum-free Stem-Pro culture media (Gibco) sup- potential bystander effect, however, is provided by our plemented with Stem-Pro Nutritional Supplement recent in vitro and in vivo observations demonstrating a (Gibco), interleukin-3 (IL-3, 5 ng/ml), IL-6 (10 ng/ml) direct toxic bystander effect of human CD34+ HSVtk- and stem cell factor (SCF, 25 ng/ml). All growth factors transduced cells on human endothelium and tumor cells were purchased from R&D Systems (Minneapolis, MN, (J Go´mez-Navarro and W Arafat, unpublished data). USA). Cells were incubated at 1 × 106 cells per well in a These facts establish a compelling rationale for our cur- 12-well flat bottom plate. Herpes virus stock was directly rent attempts at trying to define the potential role of added to the cultures at 0, 0.3, 3 and 30 multiplicities of CD34+-based cellular vehicles in a mouse tumor model. infection (MOI). Infections were initiated 1 h after start- The fact that our previous work was performed using ing the culture. In the case of primate CD34+ cells, culture both human and non-human primate CD34+ cells, a most was started approximately 8 h after BM extraction. Rigor- stringent fact in terms of levels of gene transfer achiev- ous aseptic technique was observed during the entire able by available vectors, emphasizes the potential feasi- procedure. At 12 h after infection, flow cytometric analy- bility for clinical application. Thus, the observations sis or autologous transplantation of primate cells was derived from our studies, we believe, have the potential performed. to offer a method for systemic gene delivery into disseminated areas of angiogenesis, potentially including grow- Cytotoxicity studies in vitro ing tumor foci. The process for obtaining autologous To evaluate the cytotoxicity of the TOZ.1 vector, a cell CD34+ cells is currently widely available, all of which survival assay was performed. Human CD34+ cells were makes the ultimate goal of applying this gene delivery plated in quadruplicate wells on 96 well plates, at 10000 and anti-vascular strategy to human gene therapy cells per well, and infected with 0, 0.3, 3 and 30 infectious eminently attractive. particles per cell in serum-free Stem-Pro media. In addition, Vero cells (African green monkey kidney cells) were also assayed. Cell viability was determined after 2, Materials and methods 4 and 7 days using a colorimetric cell proliferation assay that measures conversion of a tetrazolium salt to forma- Recombinant herpes virus vectors zan by viable cells, as described by the manufacturer To study the transduction efficiency of herpes virus vec- (Cell Titer 96 Aqueous Non-radioactive Cell Proliferation tors in CD34+ cells, we used TOZ.1, a non-replicative vec- Assay, Promega, Madison, WI, USA). Briefly, 20 ␮l assay tor encoding the reporter gene lacZ and deleted in the reagent mixture was added to each well containing 100 ␮l genes ICP4, ICP22, ICP27 and UL41, which are related to of cell suspension. The plates were incubated for 3 h at viral cytotoxicity (Figure 1). As a negative control we 37°C before absorbance was measured at 490 nm in a 96 used T3, a similar vector except for lacking the lacZ gene. well plate reader (Molecular Devices, Menlo Park, CA, The construction and preparation of these vectors, USA). To quantify cell numbers for the experimental derived from a KOS strain mutant, has been described results, a standard curve was generated by plating CD34+ elsewhere.61 Viral stocks were prepared and titered using cells in triplicate wells on the day of the assay at the fol- 7B cells, which express ICP4 and ICP27, as previously lowing concentrations: 50000, 20000, 10000, 5000, 2000,

Gene Therapy Endothelial progenitors for systemic gene delivery J Gomez-Navarro et al 50 and 0 cells per well. From the resulting standard curve, tee. The animals were maintained in a restricted-access cell numbers were calculated for the experimental groups facility and received NIH monkey chow and water using SOFTmax computer software (Molecular Devices). through an automatic water system containing an in-line 0.22 ␮m filter. Determination of transduction efficiency by flow cytometry analysis of ␤-galactosidase activity Tropism for areas of angiogenesis of gene-modified To determine transduction efficiency, either aliquots of CD34+ cells and transgene expression transduced cells or peripheral blood mononuclear cells After autologous transplantation, transduced cells, which were stained with anti-human CD34-biotin antibody should express the TOZ.1-encoded lacZ reporter gene, (CellPro) or an irrelevant antibody conjugated with strep- were sought in sites of angiogenesis by X-gal staining, as tavidin-PerCP (Becton-Dickinson, San Jose, CA, USA). follows. Wedge biopsies were obtained from skin grafts After 20 min of incubation in the dark, and then being and subcutaneous Matrigel and Gelfoam pellets of pri- washed, the cells were stained with fluorescein di-beta- mates autologously transplanted 5 days before with gen- d-galactopyranoside (FDG; Sigma, St Louis, MO, USA) etically modified CD34+ cells. The samples were snap by the FACS-gal technique of Fiering et al.59 Briefly, after frozen (−70°C) in tissue embedding media (Histo Prep; incubation in a 37°C water bath for 5 min, 50 ␮l of dis- Fisher Scientific, Pittsburgh, PA, USA). Frozen blocks tilled water containing 2 mm FDG was mixed thoroughly were sectioned, rinsed twice with PBS, and then fixed at with an equal volume of cells suspended in staining 4°C for 2 h in 0.5% glutaraldehyde. Next, sections were media (SM) (10 mm HEPES and 4% FCS in 1 × phosphate- rinsed twice at 37°C for 15 min in PBS containing 1 mm

buffered saline (PBS), pH 7.3). The cells were then MgCl2 and incubated with X-gal solution (PBS pH 7.4, returned to the water bath for exactly 1 min. FDG cell 5mm potassium ferrocyanide, 5 mm potassium ferricyan- ␮ loading was stopped by adding 500 l of ice-cold SM. ide, 1 mm MgCl2, and 1 mg/ml X-gal) at room tempera- Cells were maintained in ice until the FACS analysis was ture. After development of color, tissues were rinsed performed approximately 30 min later. Cells not incu- twice thoroughly in PBS with 3% DMSO, then three times bated with FDG served as the negative control for FDG in 70% ethanol, and cleared with a substitute of xylene fluorescence and were used to set the upper boundary of (Hemo-DE; Fisher). Sections were then counterstained the negative cell autofluorescence. Cells emitting signal with eosin or antibodies against endothelial-specific Fac- over this threshold were counted as FDG(+) cells. Flow tor VIII (Dako, Carpinteria, CA, USA), and analyzed by cytometry data acquisition was performed in the first light microscopy for the appearance of the characteristic 30 min after staining in a FACScalibur flow cytometer blue staining representing ␤-galactosidase activity. (Becton Dickinson), and analysis was performed with To confirm the presence in areas of angiogenesis of CellQuest software. cells transduced by the TOZ.1 vector, in situ hybridization was performed for transgene (lacZ) vector In vivo model of angiogenesis and general management sequences. Serial paraffin sections were mounted on glass of primates slides, deparaffinized, rinsed in 2 × standard saline citrate To evaluate the homing capacity of CD34+ cells into areas (SSC), and immersed in 3% hydrogen peroxide at 37°C of angiogenesis, we established an in vivo model based on for 10 min to quench endogenous peroxidase activity. implantation in primates of skin autografts, which should After proteinase K digestion for 15 min at 37°C, slides undergo a robust angiogenic reaction. In addition, as a were then dehydrated and air-dried, and a cover slip was versatile alternative source of study material, we applied and then heated at 95°C for 10 min to denature implanted subcutaneously, in several separate areas of viral DNA. Sections were incubated for 2 h at 50°C with the abdominal wall, pellets of either Matrigel (500 ␮l) a biotinylated probe in 50% formamide, 3 × SSC, (Becton Dickinson) or clinical grade Gelfoam (0.5 cm2) Denhardt’s solution, dextrose sulfate, and 10 mm DTT. (Upjohn, Kalamazoo, MI, USA). These materials were The probe was obtained using the following primers impregnated with 1 ␮g of the potent angiogenic factors against the lacZ gene sequence: 5Ј-TTG CTG ATT CGA vascular endothelial growth factor (VEGF; a kind gift GGG GTT AAC CGT CAC GAG and 3Ј-ACC AGA TGA from Genentech, South San Francisco, CA, USA) and TCA CAC TGC GGT GAT TAC GAT. Slides were then basic fibroblast growth factor (bFGF; Boehringer washed in 50% formamide, 2 × SSC, and 25 mm DTT at Mannheim, Indianapolis, IN, USA). 50°C for 20 min and incubated with a horseradish peroxi- Two days after skin graft and pellet implantation, 1– dase-conjugated streptavidin (Zymed Laboratories, South 2 × 106 CD34+ enriched transduced cells/kg were infused San Francisco, CA, USA). The reaction product was vis- into the saphenous vein. Normal, 3–4 kg rhesus ualized by means of the AEC chromogen. Figure 3a and macaques (Macacca mulatta), 2–3 years old, were obtained b show in situ hybridization of CD34 cytospin slides pro- from breeding colonies at LABS (Yemassee, SC, USA), the cessed without and with the biotinylated lacZ probe, University of Wisconsin (Madison, WI, USA), and Hazle- respectively. Figure 3c shows the skin graft biopsy ton (Alice, TX, USA). They were conditioned in a quaran- obtained in a rhesus monkey 2 weeks after infusion of tine facility for 60 days. Grafts, biopsies and venous HSV-infected CD34+ cells and similarly processed. In the blood sampling were performed under ketamine anes- inset, two cells positive for HSV genome are shown. Both thesia. The antibiotic cephazolin was administered seem to be located in the wall of capillary tubes. prophylactically (three doses at 12.5 mg/kg) immediately As a further means to show the functional conse- after transplantation. Surgical and non-surgical pro- quences of expression of genes delivered by CD34+ cells cedures were carried out in accordance with the pro- at the angiogenesis areas, we exploited the presence in visions of the NIH ‘Guide for the Care and Use of Labora- the TOZ. 1 herpes virus vector of the thymidine kinase tory Animals’ under the supervision of the University of gene. To this end, treatment with ganciclovir (Cytovene; Alabama at Birmingham Animal Care and Use Commit- Syntex Laboratories, Palo Alto, CA, USA) was initiated

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