Conventional Adenoviral Vectors Research Articles

A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy.

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

Novel REIC/Dkk-3-encoding adenoviral vector as a promising therapeutic agent for pancreatic cancer.

Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3), a tumor suppressor gene, is downregulated in various cancers. We previously reported the tumor-inhibitory effects of the REIC/Dkk-3 gene, delivered by a conventional adenoviral vector (Ad-CAG-REIC) in pancreatic cancer. Here, we developed an Ad-REIC vector with a novel gene expression system, termed the super gene expression (SGE) system, and assessed its therapeutic effects relative to those of Ad-CAG-REIC in pancreatic cancer cells. Human pancreatic cancer cell lines ASPC1 and MIAPaCa2 were used. REIC/Dkk-3 expression was assessed by western blot analysis. Relative cell viability and apoptotic effects were examined in vitro. The anti-tumor effects of Ad-REIC treatment were assessed in the mouse xenograft model. Compared with Ad-CAG-REIC, Ad-SGE-REIC elicited a significant increase in REIC protein expression in the cells studied. Relative to Ad-CAG-REIC, Ad-SGE-REIC reduced cell viability and induced apoptosis in the ASPC1 and MIAPaCa2 cell lines in vitro, and achieved superior tumor growth inhibition in the mouse xenograft model. Compared with conventional Ad-REIC agents, Ad-SGE-REIC provided enhanced inhibitory effects against tumor growth. Our results indicate that Ad-SGE-REIC is an innovative therapeutic tool for pancreatic cancer.

Creating a Future of Transplantation.

On behalf of the Japan Society for Organ Preservation and Medical Biology (JSOPMB), I express my sincere appreciation to Dr. David J. Eve and Professor Cesar V. Borlongan, Coeditors of Cell Medicine, for providing us with such an excellent opportunity to publish the data that were presented at the annual meeting of the JSOPMB. I also thank Dr. David J. Eve for the editing of our articles in detail. I am very sure that the relationship between Cell Medicine and JSOPMB has enhanced the motivation of JSOPMB members as well as board members and will continue to do so in the future, while also encouraging young Japanese researchers to join this organization. One of the extremely important missions of the annual meeting of the JSOPMB is to exchange new research outcomes and create new therapeutic concepts. JSOPMB always encourages and motivates young investigators. JSOPMB was started in 1974 for the study of organ preservation and developed widely in the 1990s with the participation of researchers in various fields of medicine, pharmacology, engineering, veterinary medicine, and basic science. Currently, JSOPMB has more than 700 members and is run under the direction of Professor Takashi Kondo, the president of the JSOPMB. Excellent presentations conducted at the 41th annual meeting of the JSOPMB held on November 28–29, 2014, in Osaka, Japan, under the supervision of Dr. Yoshiki Sawa (Professor, Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan), were selected and given an opportunity to be published in this special issue of Cell Medicine. Seven of these presentations are herein published in this special JSOPMB issue. Stem cell research was a major topic of interest. There were two articles regarding stem cells. Miyagi-Shiohira et al. reviewed cryopreservation of mesenchymal stem cells, especially adipose-derived stem cells. Dimethyl sulfoxide (DMSO) is commonly used as a cryopreservation medium as it diffuses into the cell through the plasma membrane and protects the cells from the damage caused by freezing. As substitutes for DMSO or animal-derived serum, cell banker series, polyvinylpyrrolidone (PVP), sericin and maltose, and methyl cellulose (MC) have been investigated for their clinical applications. Saitoh et al. evaluated feeder cells for establishing induced pluripotent stem cells (iPSCs) from human deciduous tooth dental pulp cells (HDDPCs). Their data showed that mouse embryonic fibroblasts (MEFs) were better feeder cells than an immortalized line established from Santos inbred mouse (SIM) embryonic fibroblasts resistant to 6-thioguanine and ouabain known as STO cells for establishing iPSCs. There were three articles regarding pancreatic islets. Noguchi et al. reviewed islet culture/preservation before islet transplantation. In the current clinical culture conditions, it seems that islets cultured at 37°C cannot receive enough oxygen because central necrosis of islets during culture was frequently observed. Low-temperature storage may be better than culture, although a future study should carefully evaluate the effects of islet equivalent and tissue volume in relation to the antigenicity of 4°C-preserved islets. Hanayama et al. showed efficient gene transduction of dispersed islet cells in culture using fiber-modified adenoviral vectors harboring a polylysine (K7) peptide in the C-terminus of the fiber knob. The fiber-modified adenoviral vector yielded higher transduction efficiencies than conventional adenoviral vector at a multiplicity of infection (MOI) of 5 and 10. Yamashita et al. evaluated human laminin (HL) isotype coating for creating islet cell sheet. HL-332 was an optimal human-derived extracellular matrix (ECM) for coating temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) surfaces. Miyamoto et al. showed a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be useful for application as a toxicity evaluation system for drug testing. Pillai et al. have achieved the fluorescence off state of quantum dots (QDs) by the conjugation of black hole quencher (BHQ) molecules intermediated with a peptide using streptavidin-QDs585 and biotin-pep-BHQ-1. The technology is expected to be useful as molecular imaging with on–off control of QD fluorescence. The theme of this JSOPMB issue is “Creating a Future of Transplantation.” The board members and I are looking forward to seeing further progress in JSOPMB in conjunction with Cell Medicine.

A novel gene expression system strongly enhances the anticancer effects of a REIC/Dkk-3-encoding adenoviral vector

Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.

Advances in Helper-Dependent Adenoviral Vector Research

The immunogenicity and cytotoxicity associated with early generations of adenoviral vectors provided a strong incentive for the development of helper-dependent adenovirus, a last generation of adenoviral vectors that is devoid of all viral coding sequences. These vectors have shown to mediate longer high-level transgene expression in vivo with reduced toxicity and thus offer enormous potential for human gene therapy. In addition, they possess a considerably larger cloning capacity than conventional adenoviral vectors making the transfer of large cDNAs, multiple transgenes and longer tissue-specific or regulable promoters possible. In this article, we review the progress made with helper-dependent adenoviral vectors. The development and optimization of scalable production processes and strategies for helper removal will be presented. Current chromatography options available for vector purification and the new challenges facing researchers for the separation of empty particles and/or helper viruses will be discussed. Finally, we will describe recent advances made in our understanding of their interaction with the immune system and their potential as gene delivery vehicles in vivo for the treatment of diseases affecting liver, skeletal muscle and brain.

Targeted delivery of NK4 to multiple lung tumors by bone marrow-derived mesenchymal stem cells

Most advanced solid tumors metastasize to different organs. However, no gene therapy effective for multiple tumors has yet been developed. Since a unique characteristic of bone marrow-derived mesenchymal stem cells (MSCs) is that they migrate to tumor tissues, we wanted to determine whether MSCs could serve as a vehicle of gene therapy for targeting multiple tumors. First, we confirmed that mouse MSCs preferentially migrate to multiple tumors of the lung in the Colon-26 (C-26) lung metastasis model. Next, MSCs were efficiently transduced with NK4, an antagonist of hepatocyte growth factor (HGF), by an adenoviral vector with an RGD motif. MSCs expressing NK4 (NK4-MSCs) strongly inhibited development of lung metastases in the C-26 lung metastasis model after systemic administration via a tail vein. Treatment with NK4-MSCs significantly prolonged survival of the C-26-tumor-bearing mice by inhibiting tumor-associated angiogenesis and lymphangiogenesis and inducing apoptosis of the tumor cells. MSC-based gene therapy did not induce the severe adverse effects induced by conventional adenoviral vectors. These results indicate that MSCs can serve as a vehicle of gene therapy for targeting multiple lung metastatic tumors.

Suitability of hCMV for viral gene expression in the brain

To the editor: Viral gene delivery is a powerful tool that is rapidly gaining popularity in experimental neuroscience. I believe that the near future will see rapid expansion of this remarkably versatile and adaptable method. Recent communication by Wickersham et al.1 describes yet another interesting implementation of this technology for retrograde tracing of neurons from their target areas. The key point of the paper is demonstration of a high level of enhanced GFP (EGFP) expression using a rabies virus mutant with glycoprotein gene substituted by EGFP. After injecting the vector into the thalamus, the authors showed many bright EGFPexpressing cortical pyramidal neurons. As a reference, they used a lentivirus with rabies virus envelope glycoprotein in which EGFP expression was driven by human cytomegalovirus (hCMV) promoter and found a very low level of EGFP expression with it. I would like to draw the attention of Nature Methods’ readers to the issue of suitability of hCMV-driven constructs for targeting brain neurons. According to the experience of our laboratory, hCMV expression in brain is only stable in some neuronal phenotypes, for example, cholinergic neurons such as those found in the nucleus of the hypoglossal nerve or vagal motor neurons. Other neurons, for example, in the adjacent nucleus of the solitary tract (which are glutamate, GABA and noradrenergic neurons) yield extremely poor expression with hCMV-containing adenoand lentiviruses2. Hence, the assumption that hCMV-driven viral constructs are guaranteed to express in all neurons is incorrect. Such constructs are, however, very active in glia, sometimes creating a confusing impression of high level of expression (see Supplementary Fig. 1 online). A striking example of a lack of hCMV activity in medullary neurons was recently given3. Therefore, for the comparison to be fair, it is critical that stable and high-level EGFP expression in pyramidal neurons be demonstrated using the same lentivirus (presumably this would require using viruses coated with vesicular stomatitis glycoprotein rather than rabies virus glycoprotein). Incidentally, we also had demonstrated bright unstained EGFP-expressing noradrenergic neurons targeted using conventional adenoviral vectors injected into spinal cord2. Such cells are easily identifiable in acute slices prepared from pretransfected animals and can be studied using patch clamp. Curiously, it seems that the phenotype of neurons affects their suitability for retrograde transduction, as we never succeeded in achieving the same with glutamatergic neurons, such as cortical pyramidals, which were the target in the study by Wickersham et al.1. Finally, it is likely that the expression profile of hCMV-containing vectors based on adeno-associated virus may obey different rules owing to the effects of the viral inverted terminal repeats4.

Suppressor of cytokine signaling-1 expression by infectivity-enhanced adenoviral vector inhibits IL-6-dependent proliferation of multiple myeloma cells

Multiple myeloma (MM) accounts for 10% of hematological malignant disorders. Its refractory nature indicates the necessity of developing novel therapeutic modalities. Since interleukin 6 (IL-6) is one of the major growth factors for MM cells, we expressed suppressor of cytokine signaling-1 (SOCS-1), one of the blockades of IL-6 receptor downstream signaling, to suppress the proliferation of MM cells. Because MM cells are resistant to conventional adenoviral vector infection, we utilized infectivity-enhanced adenoviral vectors with an RGD4C motif in the adenoviral fiber-knob region (RGD-modified vector). In infectivity analysis, RGD-modified vectors were superior to unmodified controls in the majority of the MM cell lines tested. The overexpression of SOCS-1 using infectivity-enhanced adenoviral vectors achieved growth suppression in IL-6-dependent MM cells, but not in the IL-6-independent cells. IL-6-induced STAT3 phosphorylation was suppressed in IL-6-dependent cells, indicating that the signal transduction cascade of the IL-6 receptor signaling was blocked. In aggregate, SOCS-1 overexpression with RGD-modified adenoviral vectors achieved the antiproliferative effect in IL-6-dependent MM cells. These results provide an initial proof-of-principle of the anticancer effect of SOCS-1 expression vector as well as a promise for the future development of therapeutic modality for MM based on this vector.

877. Long-Term Transgene Expression with a Second Generation Hybrid AdenoRetroviral Vector

Previously, we reported the construction of a prototype hybrid adenoretroviral vector, AdLTR-luc, which included Moloney murine leukemia virus (MoMLV) elements flanking (|[sim]| 2.7 kb 5|[prime]|; |[sim]| 1 kb 3|[prime]|) transgene cassette (Zheng et al., Nat. Biotech., 2000). Like traditional adenoviral vectors, this hybrid adenoretroviral vector is capable of transducing a wide variety of dividing and non-dividing cell types, but like retroviral vectors AdLTR-luc is able to integrate in the host cell genome resulting in long-term transgene expression. To further develop this unique vector, and improve production efficiency, we modified MoMLV sequences in the original hybrid vector. We report here the construction of a second generation hybrid vector, containing either of two transgenes: human erythropoietin (hEPO) or enhanced green fluorescence protein (EGFP). In addition, the transgene cassette included the human elongation factor 1 alpha (EF1a) promoter. Two resulting vectors, AdLTR2EF1a-hEPO and AdLTR2EF1a-EGFP, used 0.8 kb MoMLV elements (0.2 kb envelope and 0.6 kb LTR sequences in a 5|[prime]| to 3|[prime]|direction) upstream, and 0.6 kb LTR sequence downstream, of the transgene. The selection of these fragments was based on a series of gel shift assays following incubation of nuclear protein extracts with different MoMLV sequences. Production of both vectors was efficient and comparable to that obtained with first generation adenoviral vectors (|[sim]|1012 - 1013 particles/ml by Q-PCR). To evaluate if these constructs were able to integrate into genome, the rat submandibular gland A5 cell line was transduced with AdLTR2EF1a-EGFP at 100 particles/cell. After 10 days, colonies were counted and 8.1% (of 869) exhibited green fluorescence while none of the colonies (|[sim]| 900 each) obtained after similar transduction with two conventional adenoviral vectors, AdCMV-EGFP and AdEF1a-EGFP, exhibited green fluorescence. PCR assays on DNA from the cloned cells transduced with AdLTR2EF1a-EGFP were consistent with possible transgene integration into genomic DNA. To test if these constructs could mediate long-term transgene expression in vivo, AdLTR2EF1a-hEPO was delivered into rat submandibular glands at 109 particles/gland (only one gland targeted). Three of 5 rats showed elevated hEPO and hematocrit levels for two months, and in one rat elevated hEPO and hematocrit levels persisted until the end of the experiment (5 months). Transduction with the conventional adenoviral vectors, AdCMV-hEPO and AdEF1a-hEPO, resulted in elevated hEPO levels for |[sim]| 1 week. PCR assays indicated that after AdLTR2EF1a-hEPO transduction, the EF1a-hEPO sequence was found in extracted DNA at 5 months endpoint from targeted submandibular glands of all three rats expressing hEPO for at least two months. In two of these samples, PCR assays also suggested that an integration event occurred. We detected anti-hEPO antibody in sera from the two rats whose hEPO levels decreased after two months. However, in one rat with persistent high levels of hEPO for 5 months, there was no evidence of anti-hEPO antibody. Together, these results suggest that this second generation hybrid adenoretroviral vector, which is more efficiently produced than the original hybrid vector, can mediate long-term transgene expression in vivo.

980. Evaluation of the Tumor Specificity of a Conditionally Replicative Adenovirus Controlled by a Modified Human Core Telomerase Promoter

Background: Conditionally replicative adenoviruses (CRADs) are innovative anticancer agents with potential advantages over conventional adenoviral vectors due to their oncolytic effect and the ability to expand into the tumors. A strategy to achieve tumor specificity is to use tumor-selective promoters to control the expression of early viral genes essential for replication, such as E1A. The telomerase reverse transcriptase (hTERT) core promoter has been recently used to construct new CRADs because it is silenced in normal cells and active in 85% of malignancies such as liver, lung, gastric, breast, bladder, and prostate cancer. Further studies showed that the addition of 3 E-box motifs enhances the specificity of this sequence (255-4DEB promoter).

Integration efficiency of a hybrid adenoretroviral vector

The frequency with which the hybrid vector AdLTR-luc mediates genomic integration [Nat. Biotech. 18 (2000) 176–180] is unknown. To address this, we constructed AdLTR-red, using the AdLTR-luc backbone and the enhanced red fluorescence protein cDNA. Kinetic studies showed that AdLTR-red and a conventional adenoviral vector, AdCMV-red, entered and transduced epithelial cells comparably. AdLTR-red integration frequency in vitro, i.e., the percentage of red clones after 10–12 doubling times from limiting dilutions, was 8.0% (36/450; at 67 particles/cell). With AdCMV-red, 0/549 clones were integration-positive. Seven days after AdLTR-luc or AdCMV-luc (10 11 particles) femoral vein administration to adult rats splenocytes were prepared, stimulated with concanavalin A, and examined by FISH. AdLTR-luc integration occurred in 5–11% of mitotic rat splenocytes, while no unequivocal integration was found with AdCMV-luc. These data provide the first quantitative evidence of the frequency for genomic integration with this hybrid vector.

Reduced inflammatory reactions to the inoculation of helper-dependent adenoviral vectors in traumatically injured rat brain.

Traumatic brain injury (TBI) causes delayed neuronal deficits that in principle could be prevented by timely intervention with therapeutic genes. However, appropriate vectors for gene transfer to the brain with TBI remain to be developed. First-generation adenoviruses (fgAd) are usually associated with inflammatory and toxic effects when inoculated into brains, despite their high efficiency of gene transfer to these tissues. In this study the authors attempted to determine whether a less immunogenic gene-transfer protocol can be established in the traumatically injured rat brain using helper-dependent adenoviruses (hdAd), a novel adenoviral construct with full deletion of viral coding sequences. Their results show that transgene expression from intrahippocampally inoculated hdAd is maintained for at least 2 months after TBI, in contrast to the much shorter duration of fgAd-mediated gene expression. There was only minimal secretion of proinflammatory IL-1beta and TNF-alpha after inoculation of hdAd. Furthermore, the hdAd-mediated gene expression was associated with less microglial proliferation, astrocytic activation, and macrophage infiltration than observed in fgAd-inoculated brains. There was no additional tissue loss after hdAd inoculation compared with PBS injection. Although both anti-adenoviral and neutralizing antibodies were found in serum after brain inoculation of hdAd, they did not appear to affect transgene expression. The results suggest that hdAd are less immunogenic vectors than conventional adenoviral vectors, and offer improved vehicles for long-term therapeutic transgene transfer to traumatically injured brains.

A tropism-modified adenoviral vector increased the effectiveness of gene therapy for arthritis.

Adenoviral vectors (AdV) are used for anti-inflammatory cytokine therapy in experimental arthritis. Cell entry of AdV is dependent on the initial recognition of the coxsackie-adenovirus receptor (CAR) on cells. Recently, an Arg-Gly-Asp (RGD) motif was introduced in the HI loop of the fiber knob, this enables the adenovirus to bypass CAR and mediate cell entry via RGD binding integrins. In this study, we explored the transduction efficiency of the RGD-modified adenovirus in synovium and compared the RGD-modified with the conventional adenoviral vector for their effectiveness to modulate the murine collagen-induced arthritis (CIA) model when used to overexpress mIL-1Ra in the knee joint. Twenty-four hours after intra-articular injection of 10(7) fluorescent forming units (ffu) virus, luciferase (luc) activity in Ad5LucRGD-injected joints was up to 38 times higher than in AdCMVLuc-injected joints, and in arthritic joints the transduction efficiency was up to 69 times higher for the Ad5LucRGD viruses. Transduction of the synovial lining by the RGD-modified adenovirus containing the mIL-1Ra transgene, markedly improved the inhibition of CIA compared with the conventional virus in both a prophylactic and therapeutic treatment protocol. These results show that targeting integrins with the RGD-modified AdV improved the outcome of gene therapy for arthritis.

Prolonged transgene expression mediated by a helper-dependent adenoviral vector (hdAd) in the central nervous system.

Conventional adenoviral vectors such as E1-deleted first-generation adenovirus (fgAd) elicit striking host immune response, resulting in limited expression of the transgene. A recently described helper-dependent, or gutless, adenoviral vector (hdAd) can promote stable transgene expression in peripheral organs, including the liver. We therefore investigated the safety and durability of hdAd-mediated gene transfer to the central nervous system (CNS) of rats compared with gene delivery by fgAd. Equal amounts of either fgAd or hdAd carrying the beta geo transgene were stereotactically injected into the right hippocampus of adult rats. Transgene expression was assessed by histochemical staining, transgene stability by PCR analysis, and immune infiltration of T lymphocytes and macrophages by immunocytochemical methods. Strong transgene expression from either vector was detected in brain tissue examined on day 6 postinoculation. Thereafter, fgAd-mediated gene expression rapidly decreased, becoming undetectable by day 66, while expression from the hdAd vector persisted throughout the test period. PCR confirmed the presence of hdAd-associated DNA at 66 days postinoculation. The hdAd injection elicited apparently lower numbers of brain-infiltrating macrophages and T cells than did administration of fgAd. These results indicate improved transgene expression and reduced immunogenicity with use of hdAd to deliver genes to the CNS.

Genetic manipulation of primitive leukemic and normal hematopoietic cells using a novel method of adenovirus-mediated gene transfer.

Gene transfer into early hematopoietic cells has been problematic due to the quiescent nature of primitive cells and the lack of gene transfer vehicles with high efficiency for hematopoietic cell types. Previously, we have shown that adenoviral vectors can be used for the transduction of normal human progenitors with gene transfer efficiencies of approximately 30%. However, this approach is limited by relatively slow uptake kinetics (24-48 h) and a strong dependence on the presence of exogenous cytokines. Thus, we have modified this approach by combining adenoviral vectors with polycations to generate a virus-polycation complex, or VPC. Vehicles of this nature, when composed of conventional adenoviral vectors and polyamidoamine dendrimers, are a highly efficient means of transducing both normal and acute myelogenous leukemia (AML) cells. Moreover, the kinetics of gene transfer are markedly increased using the VPC strategy, with approximately 70% of transduction complete within 2 h. In this study, using viruses that encode green fluorescence protein (GFP), or the T cell costimulatory molecule B7.1 (CD80), we show that VPC-mediated gene transfer is an effective means of transducing normal and AML cells, including those with a highly primitive phenotype. Our data suggest that transient genetic manipulation of primitive hematopoietic cells can readily be achieved and should therefore permit a variety of research and clinical endeavors.

Adenovirus-mediated expresssion of the murine ecotropic receptor facilitates transduction of human hematopoietic cells with an ecotropic retroviral vector.

One factor limiting the ability to modify human repopulating hematopoietic cells genetically with retroviral vectors is the relatively low expression of the cognate viral receptor. We have tested sequential transduction of human hematopoietic cells with an adenoviral vector encoding the ecotropic retroviral receptor followed by transduction with an ecotropic retroviral vector. Adenoviral transduction of K562 erythroleukemia cells was highly efficiently with >95% of cells expressing the ecotropic receptor at a multiplicity of infection (MOI) of 103with a correspondingly high transduction with a retroviral vector. Ecotropic receptor expression in CD34+ cells following transduction with adenoviral vectors was increased by at least two-fold (from 20 to 48%) by replacing the RSV promoter with the CMV E1a promoter, resulting in a parallel increase in retroviral transduction efficiency. Replacing the head portion of the fiber protein in conventional adenoviral vectors (serotype 5) with the corresponding portion from an adenoviral 3 serotype resulted in ecotropic receptor expression in 60% of CD34+ cells at an MOI of 104 and a retroviral transduction of 60% of hematopoietic clonogenic progenitors. The sequential transduction strategy also resulted in efficient transduction of the primitive CD34+CD38- subset suggesting that it may hold promise for genetic modification of human hematopoietic stem cells.