Murine Leukemia Virus-based Vector Research Articles

PATZ1 is required for efficient HIV-1 infection

Successful HIV-1 infection and subsequent replication deeply depend on how the virus usurps the host cell machinery. Identification and functional characterization of these host factors may represent a critical strategy for developing novel anti-HIV-1 therapy. Here, expression cloning with a cDNA expression library identified as an inhibitor of HIV-1 infection, a carboxy-terminally truncated form of human POZ/BTB and AT-hook- containing Zinc finger protein 1 (PATZ1), a transcriptional regulatory factor implicated in development and cancer. Knockdown or knockout of endogenous PATZ1 revealed a supportive role of PATZ1 in HIV-1 infection, but not in transduction with murine leukemia virus-based retroviral vector. More specifically, knockdown or knockout of PATZ1 impaired the viral cDNA synthesis but not the entry process and expression of two PATZ1 isoforms in PATZ1-KO cells restored susceptibility to HIV-1 infection. These results indicate that PATZ1 plays an important role in HIV-1 infection.

Differences in the internalization of self-inactivating VSVG-pseudotyped murine leukemia virus-based vectors in human and murine cells

Self-inactivating VSVG-pseudotyped murine leukemia virus (SIN-VSVG-MLV) has been widely used to generate stable cell lines and produce gene delivery vectors. Despite the broad cellular tropism of the VSVG-pseudotyped MLV, we observed differential viral transduction efficiency depending on the host cell type used. In order to determine the mechanism underlying these differences, we used a GFP-expressing SIN-VSVG-MLV and analyzed the major steps of viral transduction in different cell lines including human epithelial, T-lymphocytes, monocytes and murine fibroblast cells.We observed the better transduction efficiency in HeLa cells, which was 20-fold higher than THP-1 and NIH/3T3 cells. To quantify viral internalization, we determined genomic RNA content by quantifying the early reverse transcription product. Genomic RNA and transduction levels were correlated with HeLa cells showing the higher amount of early RT product followed by tsA201 cells, while NIH/3T3, Jurkat and THP-1 had the lowest amounts. Similar results were observed when the late reverse transcription product was analyzed. Reverse transcription efficiency was 66–85% in HeLa cells and about 30% in tsA201, NIH/3T3, Jurkat and THP-1 cells. Viral integration, determined by Alu-Nested-qPCR, was higher for HeLa and lowerst for Jurkat and THP-1 cells. Interestingly, we observed that viral entry was correlated with the cellular availability of clathrin-mediated endocytosis, which was higher in HeLa and tsA201 cells, potentially explaining the higher rates of SIN-VSVG-MLV transduction and early RT synthesis observed in these cell lines.In conclusion, the SIN-VSVG-MLV vector showed significantly different rates of infectivity depending on the host cell type, possibly due to differential rates of viral internalization.

Human SMOOTHENED inhibits human immunodeficiency virus type 1 infection

Human SMOOTHENED (SMO) was identified by expression cloning as a new host factor that inhibits HIV-1 infection. Forced expression of SMO inhibited HIV-1 replication and infection with a single-round lentiviral vector, but not infection with a murine leukemia virus-based retroviral vector in human MT-4 T cells. Quantitative PCR analyses revealed that stable expression of SMO impaired formation of the integrated form of lentiviral DNA, but did not interrupt reverse transcription. This inhibition was evident in MT-4 and HUT102 human T cell lines expressing low levels of SMO mRNA, but not in SupT1 or Jurkat T cell lines expressing higher levels of SMO mRNA. Depletion of SMO mRNA in Jurkat cells facilitated HIV-1 vector infection, suggesting that endogenous SMO plays a role in limiting lentiviral infection. These results suggest that SMO inhibits HIV-1 replication after completion of reverse transcription but before integration.

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs. The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Amyloid β Is Not the Major Factor Accounting for Impaired Adult Hippocampal Neurogenesis in Mice Overexpressing Amyloid Precursor Protein.

SummaryAdult hippocampal neurogenesis was impaired in several Alzheimer's disease models overexpressing mutant human amyloid precursor protein (hAPP). However, the effects of wild-type hAPP on adult neurogenesis and whether the impaired adult hippocampal neurogenesis was caused by amyloid β (Aβ) or APP remained unclear. Here, we found that neurogenesis was impaired in the dentate gyrus (DG) of adult mice overexpressing wild-type hAPP (hAPP-I5) compared with controls. However, the adult hippocampal neurogenesis was more severely impaired in hAPP-I5 than that in hAPP-J20 mice, which express similar levels of hAPP mRNA but much higher levels of Aβ. Furthermore, reducing Aβ levels did not affect the number of doublecortin-positive cells in the DG of hAPP-J20 mice. Our results suggested that hAPP was more likely an important factor inhibiting adult neurogenesis, and Aβ was not the major factor affecting neurogenesis in the adult hippocampus of hAPP mice.

Retroviral Vectors Induce Epigenetic Chromatin Modifications and IL-10 Production in Transduced B Cells via Activation of Toll-like Receptor 2

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2(d)) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor [mouse cationic amino acid transporter 1 (mCAT1)] and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-κB (NF-κB) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination.

Development of murine leukemia virus-based retroviral vectors with a minimum possibility of cis-activation

The possibility of insertional mutagenesis in retroviral gene therapy can be reduced by using a vector lacking the enhancer sequence present in the U3 of the long-terminal repeats. However, such vectors suffer from many pitfalls. We attempted to improve a murine leukemia virus-based retroviral vector containing the enhancer-free U3, first by making it easier to construct a producer line and then by introducing the cellular RPL10 promoter as an internal promoter. The reverse orientation of the expression cassette of the transgene was found to give higher transducing titer and higher-level gene expression. The deletion analysis revealed that the 54-bp-long sequence of U3 (34 and 20 bp present at 5' and 3' extreme ends, respectively) was sufficient for the functions of retroviral vectors. The data from the in vitro cell culture assay indicated that the final construct, ROK, containing all these features, had little cis-activation activity, even if it was placed right upstream from the RNA start site of the neighboring gene. Our data suggested that the newly developed vector might provide increased safety, while still producing high viral titer from a stable producer line and high-level gene expression in various target cells including human CD34(+) stem cells.

Stem cells, ribozymes and HIV

In a recent Phase 2 clinical trial, Mitsuyasu et al. demonstrated that the delivery of an anti-HIV ribozyme from a gammaretroviral vector integrated into hematopoietic stem cells (HSCs) offers a safe and promising gene therapy strategy for the treatment of this infectious disease. This gene therapy strategy relies on the idea that, after the ex vivo transduction of an anti-HIV gene into autologous CD34+ HSCs, these human immunodeficiency virus (HIV)-resistant progenitors can be re-infused into an HIV patient, where they will differentiate and expand to permanently protect against the onset of AIDS (Figure 1). Although the inhibition of HIV has been considered a feasible and attractive candidate for gene therapy applications for over 20 years, this is the first reported randomized, double-blind cell-delivered Phase 2 clinical trial for HIV-1. The Mitsuyasu et al. study monitored 74 HIV-1-infected individuals for 100 weeks after infusion of transduced or control autologous HSCs. Thirty-eight of these patients were treated with HSCs carrying an integrated copy of a therapeutic, replication-incompetent, Moloney murine leukemia virus-based gammaretroviral vector (LNL6). The therapeutic vector (OZ1) expresses a hammerhead ribozyme against overlapping reading frames of the viral genes vpr and tat in unspliced and spliced viral transcripts, respectively. Although the researchers observed no significant differences in the viral load between OZ1 and control groups at any particular time point, they reported that transduced CD34+ cell therapy did not cause any apparent adverse effects and is safe for further exploration, thereby opening the doors for further trials with other gene therapy agents. Over the course of the 100-week clinical study, Mitsuyasu et al. observed sharp declines in the percentages of patients who maintained detectable levels of integrated DNA and mRNA from the OZ1 retroviral vector. In fact, at 20 weeks post-infusion, primary blood mononuclear cells from more than half of the treated patients had no detectable levels of OZ1 DNA or mRNA. This result is unexpected under the assumption that the OZ1-transduced HSCs successfully engrafted, differentiated and expanded into the T-cell lineage, and maintained some level of anti-HIV protection over untreated CD4+ T cells. Similar Phase I trials—including reports from the same authors—have confirmed successful engraftment, differentiation and expansion of autologous HSCs transduced with anti-HIV retroviral vectors. Therefore, although the HSCs may have failed to engraft under the conditions of the trial, it is also possible that the anti-HIV ribozyme failed to protect the OZ1 cells from HIV-1 infection and replication. This could be the result of mutational escape of the virus, suboptimal function of the ribozyme in vivo or possible shortcomings associated with gammaretroviral vectors. This Phase 2 investigation marks an important step toward a safe, cell-delivered gene therapy for HIV. However, to further advance this promising gene therapy approach, it will be necessary to identify the particular limitations and modify the existing therapeutic strategy. First, the virus might have acquired mutations in the target region of the ribozyme that would abrogate the function of the anti-HIV agent. Mutations arising at the second or third positions of the three-nucleotide ribozyme cleavage site of spliced and unspliced viral transcripts might render the anti-HIV ribozyme ineffective. Close examination of the targeted region suggests

304 PRODUCTION OF TRANSGENIC CHICKENS EXPRESSING HUMAN ERYTHROPOIETIN IN A TETRACYCLINE-INDUCIBLE MANNER

The use of livestock animals as bioreactors to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to agricultural improvement and medical advancement. We report here the production of transgenic chickens that produce human erythropoietin (hEPO) using replication-defective Moloney murine leukemia virus-based vectors. Because it is well known that constitutive overexpression of some cytokine genes in the transgenic animals may cause serious physiological disturbances, the vectors were designed to express in the presence of tetracycline. In addition, we introduced woodchuck hepatitis virus posttranscriptional regulatory element sequence at 3′ end of hEPO gene to boost the gene expression under the inducible condition. Approximately 5 μL of vector virus solution concentrated as much as 109 cfu mL–1 was injected beneath the blastoderm of non-incubated chicken embryo (stage X). Out of 596 injected eggs, 36 chicks hatched after 21 days of incubation and 23 hatched chicks were found to express vector-encoded hG-GSF gene when fed with doxycycline. Quantitative analysis of the blood samples taken from some Go transgenic chickens resulted in more than 300 IU mL–1 of hEPO in the blood. These transgenic chickens have not exhibited any physiological abnormalities; therefore, it is possible that this controllable gene expression system may be useful in minimizing detrimental side effects when used to produce other transgenic animals. The biological activity of the recombinant hEPO was comparable to its commercially derived Escherichia coli counterpart. The significance of this work stems from the fact that it is the first successful report on the production of transgenic chickens expressing the hEPO gene. This approach can be employed to create a useful transgenic model system for further studies on the chicken embryo development and the efficient production of transgenic chickens as bioreactors. This work was supported by: The BioGreen 21 Program of the Rural Development Administration, Republic of Korea; The SRC/ERC program of MOST/KOSEF (grant no. R11-2002-100-04005-0); The 2006–2011 Technology Development Program for Agriculture and Forestry (TDPAF), Ministry of Agriculture and Forestry, Republic of Korea.

Differential Dephosphorylation of the FcRγ Immunoreceptor Tyrosine-based Activation Motif Tyrosines with Dissimilar Potential for Activating Syk

The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.

Inhibition of west nile virus replication by retrovirus‐delivered small interfering RNA in human neuroblastoma cells

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.

Retroviral‐based gene therapy with cyclooxygenase‐2 promotes the union of bony callus tissues and accelerates fracture healing in the rat

An in vivo gene therapy strategy was developed to accelerate bone fracture repair. Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3'-untranslated region and to improve protein translation. In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E(2) and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed. This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair.

Development of Transgenic Chickens Expressing Human Parathormone Under the Control of a Ubiquitous Promoter by Using a Retrovirus Vector System

Transgenic chickens, ubiquitously expressing a human protein, could be a very useful model system for studying the role of human proteins in embryonic development as well as for efficiently producing pharmaceutical drugs as bioreactors. Human parathormone (hPTH) secreted from parathyroid glands plays a significant role in calcium homeostasis and is an important therapeutic agent for the treatment of osteoporosis in humans. Here, by using a robust replication-defective Moloney murine leukemia virus-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein, we generated transgenic chickens expressing hPTH under the control of a ubiquitous Rous sarcoma virus promoter. The recombinant retrovirus was injected into the subgerminal cavity of freshly laid eggs at the blastodermal stage. After 21 d of incubation, 42 chicks hatched from 473 retrovirus-injected eggs. All 42 living chicks were found to express the vector-encoded hPTH gene in diverse organs, as revealed by PCR and reverse transcription-PCR analysis by using primer pairs specific for hPTH. Four days after hatching, 6 chicks died and 14 chicks showed phenotypic deformities. At 18 wk of age, only 3 G0 chickens survived. They also released the hPTH hormone in their blood and transmitted the hPTH gene to G1 embryos. However, although the embryos were alive at d 18 of incubation, none hatched. An electrochemiluminescence immunoassay further showed that the hPTH expression level was markedly elevated in mammalian cells infected by the retrovirus vector. Thus, we demonstrated that transgenic chickens, expressing a human protein under the control of a ubiquitous promoter, not only could be an efficient bioreactor for the production of pharmaceutical drugs, but also could be useful for studies on the role of human proteins in embryonic development. To our knowledge, this is the first report on the production of a human protein (hPTH) in transgenic chickens under the control of a ubiquitous promoter by using a replication-defective Moloney murine leukemia virus-based retrovirus vector system.

Effects of Viral Strain, Transgene Position, and Target Cell Type on Replication Kinetics, Genomic Stability, and Transgene Expression of Replication-Competent Murine Leukemia Virus-Based Vectors

The limited efficiency of in vivo gene transfer by replication-deficient retroviral vectors remains an obstacle to achieving effective gene therapy for solid tumors. One approach to circumvent this problem is the use of replication-competent retroviral vectors. However, the application of such vectors is at a comparatively early stage and the effects which virus strain, transgene cassette position, and target cell can exert on vector spread kinetics, genomic stability, and transgene expression levels remain to be fully elucidated. Thus, in this study a panel of vectors allowing the investigation of different design features on an otherwise genetically identical background were analyzed with respect to these readout parameters in cultures of both murine and human cells and in preformed tumors in nude mice. The obtained data revealed that (i) Moloney murine leukemia virus (Mo-MLV)-based vectors spread with faster kinetics, drive higher levels of transgene expression, and are more stable than equivalent Akv-MLV-based vectors; (ii) vectors containing the transgene cassette directly downstream of the envelope gene are genomically more stable than those containing it within the 3'-long terminal repeat U3 region; and (iii) the genomic stability of both strains seems to be cell line dependent.

Retroviral Vectors Encoding a Reverse Transcription-activated Transgene Efficiently Limit Expression of the Gene to Target Cells

Recombinant retroviral vectors are indispensable tools for the study of gene function and for therapeutic gene transfer owing to their ability to transfer and stably express foreign genes in target cells. A limitation of these vectors, however, is the difficulty in generating stable vector producer cell (VPC) lines when the vectors encode cytotoxic proteins. We developed a series of Moloney murine leukemia virus-based vectors encoding a reverse transcription-activated transgene. These vectors preclude gene expression in the producer cells, yet allow lines for transgene expression in target cells. The vectors were generated by cloning the gene of interest in reverse orientation either just upstream of the viral 3' long terminal repeat (LTR) or in the U3 region of the 3'LTR. An exogenous promoter was inserted, also in reverse orientation, at the R-U5 border of the viral 5'LTR. Upon transduction of target cells, the inserted promoter is copied to the 3'LTR during reverse transcription of the vector genomic RNA, where it then drives transgene expression. We tested this system using a green fluorescent protein (GFP) gene and the SV40 promoter. Reverse transcription-activated retroviral vectors may allow for the generation of stable retroviral VPC lines encoding cytotoxic or inhibitory genes.

Endothelial Progenitor and Mesenchymal Stem Cell–Derived Cells Persist in Tissue-Engineered Patch In Vivo: Application of Green and Red Fluorescent Protein–Expressing Retroviral Vector

An unresolved question regarding tissue-engineered (TE) cardiac valves and vessels is the fate of the transplanted cells in vivo. We have developed a strategy to track the anatomic location of seeded cells within TE constructs and neighboring tissues using a retroviral vector system encoding green and red fluorescent proteins (GFPs and RFPs, respectively) in ovine circulating endothelial progenitor cells (EPCs) and bone marrow-derived mesenchymal stem cells (BMSCs). We demonstrate that stable transduction ex vivo with high-titer Moloney murine leukemia virus-based retroviral vector yields transduction efficiency of greater than 97% GFP(+) EPC- and RFP(+) mesenchymal stem cell (MSC)-derived cells. Cellular phenotype and transgene expression were also maintained through 25 subsequent passages. Using a retroviral vector system to distinguish our pre-seeded cells from tissue-resident progenitor cells and circulating endothelial and marrow-derived precursors, we simultaneously co-seeded 2 x 10(6) GFP(+) EPCs and 2 x 10(5) RFP(+) MSCs onto the TE patches. In a series of ovine pulmonary artery patch augmentation studies, transplanted GFP(+) EPC- and RFP(+) MSC-derived cells persisted within the TE patch 7 to 14 days after implantation, as identified using immunofluorescence. Analysis showed 81% luminal coverage of the TE patches before implantation with transduced cells, increasing to 96% at day 7 and decreasing to 67% at day 14 post-implantation. This suggests a temporal association between retroviral expression of progenitor cells and mediating effects of these cells on the physiological remodeling and maturation of the TE constructs. To our knowledge, this is the first cardiovascular tissue-engineering in vivo study using a double-labeling method to demonstrate a direct evidence of the source, persistence, and incorporation into a TE vascular patch of co-cultured and simultaneously pre-seeded adult progenitor cells.

Promyelocytic Leukemia Activates Chk2 by Mediating Chk2 Autophosphorylation

Chk2 is a kinase critical for DNA damage-induced apoptosis and is considered a tumor suppressor. Chk2 is essential for p53 transcriptional and apoptotic activities. Although mutations of p53 are present in more than half of all tumors, mutations of Chk2 in cancers are rare, suggesting that Chk2 may be inactivated by unknown alternative mechanisms. Here we elucidate one such alternative mechanism regulated by PML (promyelocytic leukemia) that is involved in acute promyelocytic leukemia (APL). Although p53-inactivating mutations are extremely rare in APL, t(15;17) chromosomal translocation which fuses retinoic acid receptor (RARalpha) to PML is almost always present in APL, while the other PML allele is intact. We demonstrate that PML interacts with Chk2 and activates Chk2 by mediating its autophosphorylation step, an essential step for Chk2 activity that occurs after phosphorylation by the upstream kinase ATM (ataxia telangiectasia-mutated). PML/RARalpha in APL suppresses Chk2 by dominantly inhibiting the auto-phosphorylation step, but inactivation of PML/RARalpha with alltrans retinoic acid (ATRA) restores Chk2 autophosphorylation and activity. Thus, by fusing PML with RARalpha, the APL cells appear to have achieved functional suppression of Chk2 compromising the Chk2-p53 apoptotic pathway.

Tissue- and Tumor-Specific Targeting of Murine Leukemia Virus-Based Replication-Competent Retroviral Vectors

Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human alpha1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver- and beta-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.

The DNA repair genes XPB and XPD defend cells from retroviral infection

Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A host degradation system prevents a majority of the cDNA molecules from completing the obligatory genomic integration necessary for pathogenesis. We demonstrate that the human TFIIH complex proteins XPB (ERCC3) and XPD (ERCC2) play a principal role in the degradation of retroviral cDNA. DNA repair-deficient XPB and XPD mutant cell lines exhibited an increase in transduction efficiency by both HIV- and Moloney murine leukemia virus-based retroviral vectors. Replicating Moloney murine leukemia virus viral production was greater in XPB or XPD mutant cells but not XPA mutant cells. Quantitative PCR showed an increase in total cDNA molecules, integrated provirus, and 2LTR circles in XPB and XPD mutant cells. In the presence of a reverse transcription inhibitor, the HIV cDNA appeared more stable in mutant XPB or XPD cells. These studies implicate the nuclear DNA repair proteins XPB and XPD in a cellular defense against retroviral infection.

Maintenance of Proliferative Capacity and Retroviral Transduction Efficiency of Human Fetal CD38–/CD34++Stem Cells

Methods for the efficient transduction and expansion of fetal hematopoietic stem cells could lead to novel in utero therapies for blood cell disorders and enzymatic deficiencies. Here we describe a new assay to measure rapidly the effects of cytokines on the differentiation or expansion of primitive progenitors and stem cells found among CD38(-)CD34(++) lineage() cells isolated from human midgestation liver. Importantly, conditions that otherwise supported the expansion of clonogenic progenitors reduced their proliferative capacity. A combination of megakaryocyte growth and development factor and granulocyte-macrophage colony-stimulating factor maintained proliferative potential while also yielding an intermediate level of progenitor expansion. Retroviral transduction was achieved using Moloney murine leukemia virus-based vectors. Freshly isolated candidate stem cells could be transduced at almost 17% efficiency by a 1-h exposure to virus with centrifugation to aid transduction. This was increased to a mean 35.5% transduction efficiency after 1 day of culture. Additionally, the transduction efficiency of candidate stem cells isolated from fetal placental blood was 33.0%. These findings encourage further investigation into the feasibility of ex utero gene therapy whereby fetal cells are isolated from the circulation, transduced, and expanded ex utero before being returned to the fetus.