Transduction Of Hematopoietic Cells Research Articles

Screening of V617F mutation in JAK2 gene with acute myeloid leukemia in the Saudi population.

Progress in pathogenesis and therapy of acute myeloid leukemia (AML) is presently accelerating. The Janus kinase 2 gene (JAK2) mutations are rare in de novo AML. The gene codes for the tyrosine kinase that has a significant role in the signal transduction in hematopoietic cells. The aim of this study was to induce V617F mutation in the JAK2 gene in the AML patients diagnosed in the Saudi population. In this case-control study, 100 AML patients and 100 healthy controls were recruited. Genotyping was performed with polymerase chain reaction followed with restriction fragment length polymorphism analysis. The mean age of the AML patients and healthy controls was found to be almost similar (p=0.60). In this study, 15% of VF mutation was documented in the AML cases and none of the mutations were documented either in FF mutation in AML cases or VF and FF mutations in the healthy control subjects. VF mutations [VF vs VV; OR-18.79; (95%CIs: 2.442-144.6) and p=0.0001; F vs V; OR-87.76; (95% CIs: 11.76-654.7) and p<0.0001] were found to be significantly associated when compared between AML cases and healthy controls. In conclusion, the V617F mutation showed the positive association in the AML patients diagnosed in the Saudi population.

Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold, respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50- to 100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.

Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno-associated virus serotype 6 vectors.

Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno-associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well-known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood-derived CD34+HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype-dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector-mediated transgene expression. Taken together, these studies expand our understanding of rAAV6-mediated transduction in hematopoietic cells and are informative for improving rAAV6-based treatment of blood diseases.

Mechanisms Underlying the Anti-inflammatory and Immunosuppressive Activity of Ruxolitinib.

The JAK-STAT signaling pathway plays a central role in signal transduction in hematopoietic cells, as well as in cells of the immune system. The occurrence in most patients affected by myeloproliferative neoplasms (MPNs) of driver mutations resulting in the constitutive activation of JAK2-dependent signaling identified the deregulated JAK-STAT signal transduction pathway as the major pathogenic mechanism of MPNs. It also prompted the development of targeted drugs for MPNs. Ruxolitinib is a potent and selective oral inhibitor of both JAK2 and JAK1 protein kinases. Its use in patients with myelofibrosis is associated with a substantial reduction in spleen volume, attenuation of symptoms and decreased mortality. With growing clinical experience, concerns about infectious complications, and increased risk of B-cell lymphoma, presumably caused by the effects of JAK1/2 inhibition on immune response and immunosurveillance, have been raised. Evidence shows that ruxolitinib exerts potent anti-inflammatory and immunosuppressive effects. Cellular targets of ruxolitinib include various components of both the innate and adaptive immune system, such as natural killer cells, dendritic cells, T helper, and regulatory T cells. On the other hand, immunomodulatory properties have proven beneficial in some instances, as highlighted by the successful use of ruxolitinib in corticosteroid-resistant graft vs. host disease. The objective of this article is to provide an overview of published evidence addressing the key question of the mechanisms underlying ruxolitinib-induced immunosuppression.

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor.

Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

BCR\u2013ABL1-induced downregulation of WASP in chronic myeloid leukemia involves epigenetic modification and contributes to malignancy

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR–ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR–ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott–Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR–ABL1 and is particularly low in blast crisis. Enforced expression of BCR–ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR–ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR–ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR–ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).

99. A Novel Mechanism of Transduction of Human Cells By Recombinant AAV Vectors

The human embryonic kidney cell line, HEK293, is readily transduced by the most commonly used AAV2 serotype vectors because these adherent cells abundantly express the cell surface receptor, heparan sulfate proteoglycan (HSPG), and one of the co-receptors, human fibroblast growth factor receptor 1 (FGFR1) for AAV2. However, a human erythroleukemia cell line, K562, commonly used as a model for human hematopoietic cell transduction, is not transduced as efficiently, although these cells grown in suspension, express both HSPG and FGFR1 only at modestly lower levels. We reasoned that the lack of proximity of HSPG and FGFR1 on K562 cells might account for the suboptimal transduction of these cells, and hypothesized that if the transduction was performed at high cell density, presumably allowing for HSPG on one cell to come in close proximity to FGFR1 on the neighboring cell, then AAV2 bound to HSPG on one cell could utilize FGFR1 on the neighboring cell to gain entry in the latter, and vice versa, thus leading to increased transduction. To test this hypothesis, HEK293 and K562 cells were transduced with 3×103 vgs/cell of scAAV2-EGFP vectors either at low-density (60,000 cells in 50 ml) or at high-density (480,000 cells in 50 ml). Transgene expression was evaluated by flow cytometry 48 hrs post-transduction. Whereas ~90% of HEK293 cells expressed the transgene at low-density, the transduction efficiency at high-density increased up to ~95%. On the other hand, only ~35% of K562 cells were transduced at low-density, but interestingly, the transduction efficiency at high-density increased to ~65%, thus corroborating that close cell-cell contact facilitated vector entry into these cells. That the initial cell-cell contact was critical in achieving improved transduction, was further corroborated by experiments in which cells were transduced at low-density, and subsequently pooled together to reach high-density, and conversely, cells were transduced at high-density, and soon after transduction, were diluted to low-density. The increased transduction was observed only under the latter condition. Similar results were obtained with two additional human cell lines, M07e and Raji, which express low to extremely low levels of HSPG and FGFR1, respectively, and consequently, are transduced extremely poorly by AAV2 vectors. These studies were also extended to include primary human CD34+ hematopoietic stem/progenitor cells (HSPCs), also grown in suspension, and known to be transduced sub-optimally by AAV2 vectors. In these studies also, whereas only~5% of these cells were transduced at low-density, the transduction efficiency increased up to ~20% at high-density. Taken together, our studies have revealed a novel mechanism, which we have termed “cross-transduction”, which AAV vectors exploit to gain entry into target cells, and which may have implications in the optimal use of AAV vectors in human gene therapy applications.

100. Multifunctional Drug Enhances rAAV-Mediated Transgene Expression Through Different Mechanisms in Different Cell Types

Several groups have demonstrated that a number of naturally occurring compounds can significantly enhance the therapeutic efficacy of viral vectors, especially recombinant AAV vectors (Ren et al., Neurosci Lett. 479(3): 187-91, 2010; Zhang et al., Gene Ther. 18(2): 128-34, 2011; Mitchell et al., J Virol. 87(8): 4571-83, 2013; Wang et al., J Integr Med. 12(1): 20-34, 2014). In our recently published studies (Ling et al., Hum Gene Ther. 25(12): 1023-34, 2014), we provided further evidence of enhanced efficacy of a combinatorial approach involving drug-mediated chemotherapy and rAAV vector-mediated cancer gene therapy to target human hepatocellular carcinoma. In these studies, we identified that shikonin, a naphthoquinone, inhibits human liver tumor growth as well as significantly enhances the efficacy of rAAV vectors carrying a therapeutic gene, tricosanthin (TCS), in a murine xenograft model in vivo. Although our initial mechanistic studies suggested that shikonin-mediated enhancement of rAAV vector transduction was through its proteasome inhibitory activity, which was similar to that of MG132, a specific proteasome inhibitor. However, no further increase was observed when cells were co-treated with both shikonin and MG132.Since shikonin is a multifunctional compound, we wished to evaluate its effect on rAAV vector-mediated transgene expression in additional human cell types. In a human erythroleukemia cell line, K562, frequently used as a model for hematopoietic cell transduction studies, shikonin enhanced the transduction efficiency of various rAAV serotype vectors by up to 10-fold. Surprisingly, however, MG132 had no effect in K562 cells, even at the highest concentration that did not induce cytotoxicity. Thus, in a systematic study, various proteasome and protease inhibitors were evaluated. Western blot assays for the detection of polyubiquitinated total cellular proteins were performed to confirm the effectiveness of these drugs in K562 cells. Whereas carfilzomib only had a modest effect (<2-fold enhancement), surprisingly, bortezomib significantly reduced the AAV vector-mediated transgene expression in K562 cells. E-64 and PMSF, a cysteine and a serine protease inhibitor, respectively, also had no effect. Additional studies revealed that a specific inhibitor of the reactive oxygen species (ROS) formation, blocked the enhancing effect of shikonin on rAAV vector-mediated transgene expression in K562 cells but not other cell types. Furthermore, shikonin treatment did augment rAAV vector-mediated transduction ex vivo in both mouse and human primary hematopoietic stem cells.Taken together, our studies also emphasize the need to exercise caution in interpreting rAAV vector-mediated transgene expression data among different cell types involving the use of multifunctional drugs.

Development of a novel adenovirus–alphavirus hybrid vector with RNA replicon features for malignant hematopoietic cell transduction

To improve the expression levels of transgenes in malignant hematopoietic cells, we developed a novel adenoviral-alphavirus hybrid vector Ad5/F11p-SFV-GFP that contains a Semliki Forest Virus (SFV) replicon and chimeric fibers of Ad5 and Ad11p. Ad5/F11p-SFV-GFP infected >95% of K562, U937 or Jurkat cells and 23.65% of HL-60 cells, and led to moderate Enhanced Green Fluorescent Protein (EGFP) transgene expression intensity. The infection efficiency of Ad5/F11p-SFV-GFP in primary human leukemia cells ranged from 9.34-89.63% (median, 28.58%) at a multiplicity of infection (MOI) of 100, compared with only 3.37-44.54% (median, 10.42%) in cells infected by Ad5/F11p-GFP. Importantly, Ad5/F11p-SFV-GFP led to a significantly higher transgene expression level in primary leukemia cells, as indicated by the relative fluorescence intensity, compared to cells infected with Ad5/F11p-GFP. The increased expression of EGFP in Ad5/F11p-SFV-GFP-infected cells was associated with the accumulation of abundant subgenomic mRNA. Additionally, infection of K562, U937 or Jurkat cells by Ad5/F11p-SFV-GFP was significantly inhibited by blocking CD46 receptor; however, other factors may affect the gene-transfer efficiency of Ad5/F11p-SFV-GFP in primary leukemia cells. In conclusion, we successfully developed a novel adenoviral-alphavirus hybrid vector with RNA replicon features, which represents a promising vector for gene modifications during the production of cell-based vaccines for leukemia patients.

Centrosomal targeting of Syk kinase is controlled by its catalytic activity and depends on microtubules and the dynein motor

The nonreceptor Syk kinase is detected in epithelial cells, where it acts as a tumor suppressor, in addition to its well-established role in immunoreceptor-based signal transduction in hematopoietic cells. Thus, several carcinomas and melanomas have subnormal concentrations of Syk. Although Syk is mainly localized at the plasma membrane, it is also present in centrosomes, where it is involved in the control of cell division. The mechanisms responsible for its centrosomal localization and action are unknown. We used wild-type and mutant fluorescent Syk fusion proteins in live-cell imaging (fluorescence recovery after photobleaching, total internal reflection fluorescence, and photoactivation) combined with mathematical modeling to demonstrate that Syk is actively transported to the centrosomes via the microtubules and that this transport depends on the dynein/dynactin molecular motor. Syk can only target the centrosomes if its kinase activity is intact and it is catalytically active at the centrosomes. We showed that the autophosphorylated Y130 Syk residue helps to uncouple Syk from the plasma membrane and to promote its translocation to the centrosome, suggesting that the subcellular location of Syk depends on its autophosphorylation on specific tyrosine residues. We have thus established the details of how Syk is trafficked intracellularly and found evidence that its targeting to the centrosomes is controlled by autophosphorylation.

Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP⁻/⁻ cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP⁻/⁻ Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.

P37. Epigenetic changes induced in human hematopoietic progenitor/stem cells following ex vivo lentiviral transduction

Gene therapy is a promising strategy to treat certain inherited disorders. In that context, lentiviral vectors (LV) have been used in ex vivo protocols for the stable gene-modification of hematopoietic stem cells. There are concerns about the potential genotoxicity of integrative vectors such as LV. So far, these concerns have only been envisioned as resulting from unwanted genomic insertion leading to insertional mutagenesis. At present, there has been no investigation of potential epigenetic modification induced by the procedure of gene transfer into stem cells. Epigenetic changes are known to be induced in cells following culture and stress. This prompted us to investigate if the procedure used for ex vivo lentiviral transduction which involves a period of cytokine pre-activation prior to LV infection, could induce epigenetic alterations in human CD34+ haematopoietic/progenitor cells. The expression of a panel of known epigenetic proteins was explored by immunohistochemical analysis at initiation of the treatment, post-stimulation and post-infection of umbilical cord blood CD34+ cells. To measure the patterns and levels of expression of these proteins in the cell population and to account for the heterogeneity of the CD34+ cell population, we used high throughput image analysis with the Cell Profiler Software. As expected, the cytokine pre-activation of the cells led to a heterogeneous up regulation of several proteins known to be involved in metabolic changes and stress response. Importantly, the systematic analysis of up to 30 morphological features per cell indicated that lentiviral infection itself had a distinct effect than those of cytokines. Cells infected with the LV displayed significantly different and more heterogenous phenotypic profiles compared to non-transduced cells or pre-activated only cells. These constitute the first observations to our knowledge that ex vivo lentiviral transduction of hematopoietic cells may induce epigenetic changes. Our data suggest that lentiviral transduction may represent a stress for the cell leading to an “epigenetic storm” which could induce variable deregulation of certain genes in the cells. We show that these effects could be monitored and further experiments are ongoing to evaluate the impact of different experimental conditions on such epigenetic changes.

Production of Foamy Virus Vector and Transduction of Hematopoietic Cells

Foamy viruses (FVs), or spumaviruses, are nonpathogenic retroviruses that have been developed as integrating viral vectors. This protocol presents methods for producing high-titer FV vector stocks, free of contaminating replication-competent retrovirus, to be used for transducing hematopoietic stem cells. FV vector stocks are produced by transfecting 293 cells, harvesting and filtering the culture medium, and concentrating vector virions by ultracentrifugation. The resulting stocks are free of replication-competent helper virus, as indicated by a sensitive marker rescue assay. A typical stock made from 23 10-cm dishes has a final volume of 2 mL with a titer of 10(7) to 10(8) transducing units/mL. Potential advantages of FV vectors include a lack of pathogenicity of the wild-type virus, a wide host range, stable virions that can be concentrated by centrifugation, a double-stranded DNA genome that is reverse-transcribed in the vector-producing cells, and the largest packaging capacity of any retrovirus. FV vectors are especially useful for transducing hematopoietic cells. Because hematopoietic stem cells have the ability to self-renew, proliferate, and repopulate the bone marrow after transplantation, efficient transduction of these cells offers the promise to cure many inherited and acquired diseases.

Neutrophil elastase is severely down-regulated in severe congenital neutropenia independent of ELA2 or HAX1 mutations but dependent on LEF-1

AbstractSevere congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis which follows an autosomal dominant or autosomal recessive pattern of inheritance. Genetic analyses indicate mutations in the ELA2 gene in most patients. We have identified LEF-1 as a decisive transcription factor in granulopoiesis controlling proliferation and granulocytic differentiation by direct activation of its target gene, C/EBPα. In patients with CN, the expression of LEF-1 and C/EBPα was abrogated in myeloid progenitors leading to maturation arrest of granulopoiesis. In the present study we demonstrated that ELA2 mRNA expression in myeloid progenitors and plasma protein levels of neutrophil elastase (NE) were markedly reduced in patients with CN harboring mutations in either ELA2 or HAX-1 genes. The ELA2 gene promoter is positively regulated by the direct binding of LEF-1 or C/EBPα, documenting the role of LEF1 in the diminished ELA2 expression. We found that transduction of hematopoietic cells with LEF-1 cDNA resulted in the up-regulation of ELA2/NE synthesis, whereas inhibition of LEF-1 by shRNA led to a marked reduction in the levels of ELA2/NE. LEF-1 rescue of CD34+ cells isolated from 2 patients with CN resulted in granulocytic differentiation of the cells which was in line with increased levels of functionally active ELA2/NE.

Efficient Human Hematopoietic Cell Transduction Using RD114- and GALV-Pseudotyped Retroviral Vectors Produced in Suspension and Serum-Free Media

Retroviral vectors derived from the Moloney murine leukemia virus have been used in successful and promising gene therapy clinical trials. However, platforms for their large-scale production must be further developed. As a proof of principle, we reported the generation of a packaging cell line that produces amphotropic retroviral vectors in suspension and serum-free medium (SFM). In the present study, we have constructed and characterized two retroviral packaging cell lines designed for gene transfer in hematopoietic cells. These cell lines grow in suspension and SFM, and produce high-titer RD114- and gibbon ape leukemia virus (GALV)-pseudotyped vectors for a 3-month culture period. Viral particles released are as robust during repeated freeze-thaw cycles and on thermal inactivation at 37 degrees C as their counterparts produced in cells cultured adherently with serum. We also show that RD114- and GALV-pseudotyped vectors produced in suspension and SFM efficiently transduce human lymphocytes and hematopoietic stem cells. As these retroviral packaging cell lines distinctively maintain high vector titers while growing in suspension and SFM, we conclude that these cell lines are uniquely suitable for large-scale clinical-grade vector production for late-phase clinical trials involving gene transfer into hematopoietic cells.

286. Efficient Transduction of Hematopoietic Cells Using Retroviral Vectors Produced in Suspension and Serum-Free Media

286. Efficient Transduction of Hematopoietic Cells Using Retroviral Vectors Produced in Suspension and Serum-Free Media

Interaction of penton base Arg-Gly-Asp motifs with integrins is crucial for adenovirus serotype 35 vector transduction in human hematopoietic cells

Most subgroup B adenoviruses (Ads), including adenovirus (Ad) serotype 35 (Ad35), bind to human CD46 as a receptor; however, the infection processes of subgroup B Ads following attachment to CD46 remain to be elucidated. Subgroup B Ads possess Arg-Gly-Asp (RGD) motifs in the penton base, similarly to subgroup C Ad serotypes 2 and 5. In this study, we examined the role of penton base RGD motifs in Ad35 vector-mediated transduction in human hematopoietic cells. Inhibition of interaction between integrins and the RGD motifs by divalent cation chelation and a synthetic RGD peptide reduced the transduction efficiencies of Ad35 vectors; however, the amounts of cell-associated vector DNA of Ad35 vectors at 4 or 37 degrees C were not decreased by divalent cation chelation or the RGD peptide. Mutation of penton base RGD motifs reduced the transduction efficiencies of Ad35 vectors, although the amounts of cell-associated vector DNA of Ad35 vectors at 4 or 37 degrees C were not altered by mutation of penton base RGD motifs in Ad35 vectors. Furthermore, preincubation with several types of anti-integrin antibodies significantly inhibited Ad35 vector-mediated transduction. These results suggest that interaction between integrins and penton base RGD motifs plays a crucial role in Ad35 vector-mediated transduction in hematopoietic cells, probably in the post-internalization steps.

Lentivirus Transduction of Hematopoietic Cells: Figure 1.

INTRODUCTIONEfficient transfer and sustained expression of transgenes are among the most important issues in gene delivery. The majority of hematopoietic cells are nondividing or slowly self-renewing. Thus, they are refractory to most nonviral or retroviral delivery methods. Lentiviral vectors are capable of transducing nondividing cells and maintaining long-term and sustained expression of the transgenes. They are becoming useful for many delivery protocols, such as long-term expression of short hairpin RNA (shRNA) and functional genetics. They may also have great potential in gene therapy. This protocol describes lentivirus-vector-based delivery of foreign genes to hematopoietic cells. The method is applicable to various cell types in experiments that require long-term transgene expression.

Importance of Murine Study Design for Testing Toxicity of Retroviral Vectors in Support of Phase I Trials

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMT(P140K)wc. Test mice had significant gene marking 8-12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMT(P140K), thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMT(P140K) expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.

Deficiency of SHP-1 Protein-Tyrosine Phosphatase in “Viable Motheaten” Mice Results in Retinal Degeneration

Viable motheaten mutant mice (abbreviated allele symbol me(v)) are deficient in Src-homology 2-domain phosphatase (SHP)-1, a critical negative regulator of signal transduction in hematopoietic cells. These mice exhibit immune dysfunction, hyperproliferation of myeloid cells, and regenerative anemia. This study focused on the role of SHP-1 in retinal homeostasis. Ophthalmoscopy, histology, transmission electron microscopy (TEM), electroretinography (ERG), immunohistochemistry, Western blot, bone marrow transplantation, and genetic crosses were performed for phenotypic characterization and functional studies of retinal degeneration (RD) in me(v)/me(v) mice. Fundus examinations of me(v)/me(v) mice revealed numerous, small white spots. Histologic examination demonstrated photoreceptor loss beginning at 3 weeks of age, and TEM revealed disorganization and reduction in the number of outer segments, as well as the presence of phagocytic cells in the subretinal space. Rod- and cone-mediated ERGs were abnormal. SHP-1 protein was expressed in mouse and human retinal lysates and was localized to the outer nuclear layer of the retina in me(v)/me(v) and control mice. Autoantibodies are not necessary for RD, as B-cell-deficient me(v)/me(v) Igh-6(tm1Cgn) mice had no attenuation of photoreceptor cell loss compared with age-matched me(v)/me(v) mice. Histologic examination of lungs and retinas from normal recipients of me(v)/me(v) marrow revealed the classic acidophilic macrophage pneumonia of me(v)/me(v) mice, but no retinal degeneration. me(v)/me(v) mice exhibit normal retinal development with the onset of RD at 3 weeks of age and a rapidly progressive loss of photoreceptors. These findings support the hypothesis that SHP-1 plays a critical role in retinal homeostasis.