Retroviral Producer Research Articles

Entrapment of retroviral vector producer cells in three‐dimensional alginate scaffolds for potential use in cancer gene therapy

We explored the possibility of entrapping retroviral vector producing cells (VPC) within porous 3D matrix to induce a local and sustained release of viral particles to the malignant milieu. PA317/STK, which constantly shed retroviral vectors, was used to transduce cancer cells with the herpes simplex virus thymidine kinase (HSV-tk) gene. Once HSV-tk is expressed, it preferentially phosphorylates nucleoside analog prodrugs, such as ganciclovir (GCV) and N-methanocarbathymidine (N-MCT), to their active triphosphate metabolites, which when incorporated into cellular DNA cause cell death. PA317/STK cells were seeded within 3D alginate scaffold at two different cell densities via static seeding procedure. In vitro assays determined that PA317/STK seeded at high-cell density in scaffolds maintained constant cell number, low cell leakage, and spheroid morphology with viral vector transfection activity. Postcell-seeding viral vector activity was confirmed by transfection of murine colon cancer cells (MC38) with conditioned media originated from VPC-containing scaffolds and the subsequent ability to generate N-MCT triphosphate. Preliminary in vivo transplantation of VPC-containing scaffolds into the peritoneal cavity of mice bearing intraperitoneal MC38 tumors with 2 weeks subsequent GCV administration resulted in a significantly higher survival rate relative to control groups. Our results demonstrate the feasibility of employing alginate scaffolds to efficiently entrap and support PA317/STK cells for cancer gene therapy.

Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors

BackgroundSelf-inactivating retroviral vectors (SIN) are often associated with very low titers. Promoter elements embedded within SIN designs may suppress transcription of packageable retroviral RNA which in turn results in titer reduction. We tested whether this dominant-negative effect involves histone acetylation state. We designed an MLV-derived SIN vector using the cytomegalovirus immediate early enhancer-promoter (CMVIE) as an embedded internal promoter (SINCMV) and transfected the pantropic 293GPG packaging cell line.ResultsThe SINCMV retroviral producer had uniformly very low titers (~10,000 infectious retroparticles per ml). Northern blot showed low levels of expression of retroviral mRNA in producer cells in particular that of packageable RNA transcript. Treatment of the producers with the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A reversed transcriptional suppression and resulted in an average 106.3 ± 4.6 – fold (P = 0.002) and 15.5 ± 1.3 – fold increase in titer (P = 0.008), respectively. A histone gel assay confirmed increased histone acetylation in treated producer cells.ConclusionThese results show that SIN retrovectors incorporating strong internal promoters such as CMVIE, are susceptible to transcriptional silencing and that treatment of the producer cells with HDAC inhibitors can overcome this blockade suggesting that histone deacetylation is implicated in the mechanism of transcriptional suppression.

Retrovirus producer cell line metabolism: implications on viral productivity

The production of retroviral vectors by human cell lines is still hampered by low titers making it relatively difficult to produce very large quantities of this vector of high interest for clinical gene therapy applications. Thus, to improve vector production, we studied the influence of different sugars alone or combinations of sugars on cell growth, vector titers, and metabolism of the producer cell. The use of fructose at 140 mM or a mixed medium (with glucose at 25 mM and fructose at 140 mM) improved the virus titer three- to fourfold, respectively, and the producer cell productivity by fivefold. The increase in the cell productivity was due to a 1.5-fold increase in the vector stability, the remaining increase being due to higher cell specific productivity. The increase in the productivity was associated with lower glucose oxidation and an increase in the lactate and alanine yield. In the mixed medium, an increase in fatty acids derived from the glucose was observed in parallel with a reduction of glutamate and glutamine synthesis via the tricarboxylic acid (TCA) cycle acetyl-CoA and alpha-ketoglutarate, respectively. Although the higher productivities were associated with severe changes in the glycolysis, TCA cycle, and glutaminolysis, the cell energetic status monitored by phosphocreatine and adenosine triphosphate levels was not significantly affected. The synthesis of fatty acids and phospholipids were enhanced in the fructose or mixed media and are possibly key parameters in retroviral vector production.

Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: Predictability and efficiency by transgene exchange

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.

The Viral E3 Ubiquitin Ligase mK3 Uses the Derlin/p97 Endoplasmic Reticulum-associated Degradation Pathway to Mediate Down-regulation of Major Histocompatibility Complex Class I Proteins

Ubiquitin E3 ligases are important cellular components for endoplasmic reticulum (ER)-associated degradation due to their role in substrate-specific ubiquitination, which is required for retrotranslocation (dislocation) of most unwanted proteins from the ER to the cytosol for proteasome degradation. However, our understanding of the molecular mechanisms of how E3 ligases confer substrate-specific recognition, and their role in substrate retrotranslocation is limited especially in mammalian cells. mK3 is a type III ER membrane protein encoded by murine gamma herpesvirus 68. As conferred by its N-terminal RING-CH domain, mK3 has E3 ubiquitin ligase activity. In its role as an immune evasion protein, mK3 specifically targets nascent major histocompatibility complex class I heavy chains (HC) for rapid degradation. The mechanism by which mK3 extracts HC from the ER membrane into the cytosol for proteasome-mediated degradation is unknown. Evidence is presented here that HC down-regulation by mK3 is dependent on the p97 AAA-ATPase. By contrast, the kK5 protein of Kaposi's sarcoma-associated herpesvirus is p97-independent despite the fact that it is highly homologous to mK3. mK3 protein was also found in physical association with Derlin1, an ER protein recently implicated in the retrotranslocation of HC by immune evasion protein US11, but not US2, of human cytomegalovirus. The mechanistic implications of these findings are discussed.

A Growth-suppressive Function for the c-Fes Protein-Tyrosine Kinase in Colorectal Cancer

The human c-fes locus encodes a non-receptor protein-tyrosine kinase implicated in myeloid, vascular endothelial, and neuronal cell differentiation. A recent analysis of the tyrosine kinome in colorectal cancer identified c-fes as one of only seven genes with consistent kinase domain mutations. Although four mutations were identified (M704V, R706Q, V743M, S759F), the consequences of these mutations on Fes kinase activity were not explored. To address this issue, Fes mutants with these substitutions were co-expressed with STAT3 in human 293T cells. Surprisingly, the M704V, R706Q, and V743M mutations substantially reduced Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F had little effect. These mutations had a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. We have also demonstrated for the first time that endogenous Fes is strongly expressed at the base of colonic crypts where it co-localizes with epithelial cells positive for the progenitor cell marker Musashi-1. In contrast to normal colonic epithelium, Fes expression was reduced or absent in colon tumor sections from most individuals. Fes protein levels were also low or absent in a panel of human colorectal cancer cell lines, including HT-29 and HCT 116 cells. Introduction of Fes into these lines with a recombinant retrovirus suppressed their growth in soft agar. Together, our findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer.

Potent and Specific Inhibition of Retrovirus Production by Coexpression of Multiple siRNAs Directed Against Different Regions of Viral Genomes

siRNA-mediated RNA degradation has been demonstrated to act as an antiviral system in many species. Here we describe inhibition of retrovirus production by multiple siRNAs designed to target various regions of the viral genomes. Using murine leukemia virus (MuLV) as a model, we demonstrate that the virus production can be inhibited by 77% in siLTR2 (a siRNA targeting the U3 region of MuLV) expression vector transfected cells. Coexpression of siLTR2 with siPsi2 (a siRNA targeting the 3' Psi (packaging signal sequence) results in 93% suppression of the virus production, suggesting that an increased inhibition of the virus production can be achieved by coexpression of multiple siRNAs to target different regions of the viral RNA simultaneously. Our results also indicate that not all sequences of the viral RNA are equally accessible to siRNA. We show that U3 region of MuLV is more accessible to siRNA, whereas the packaging signal sequence, especially the region adjacent to 5'LTR, is less accessible to siRNA, partly as a result of the binding of Gag precursors. Furthermore, we demonstrate that coexpression of siLTR2 with siPsi2 in virus producer cells leads to 88% knockdown of viral titer, showing the benefit of coexpression of multiple siRNAs for potent suppression of virus production in the setting of an established infection. Moreover, we demonstrate that infection of MuLV in cells that stably coexpress siLTR2 with siPsi2 diminishes by 77%. Taken together, we establish that siRNA-mediated gene silencing can suppress multiple steps of the retrovirus life cycle, offering a potential for both treating virus-associated diseases and preventing viral infection.

815. 293 FLEX a New Cell Line for Fast, Reproducible and Efficient Production ofRetroviral Gene Therapy Vectors

This work describes the establishment of a pioneering packaging cell line for the production of retroviral Gene Therapy Vectors. The development of this 293 FLEX packaging cell line was performed not only by generating a flexible exchange system for the transgene, but also, taking into account the optimal retroviral components stoichiometry, for the enhancement of the cell-specific productivity.

816. Rapid, Consistent and Scalable Production of Viral Vectors in Suspension Cells by Cotransfection of Viral Component Plasmid DNA or RNA

Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte’s proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte’s high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy. Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte’s proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte’s high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy.

473. Semliki Based Systems for the Production of Retroviral Vectors: A Safety Concern

The development of efficient and safe production procedures are an essential goal conditioning viral vectors usefulness. For retroviral vectors production, two groups have suggested that Semliki forest virus (SFV) derived systems might generate interestingly elevated yields. The system relies on the transfection of three complementary SFV replicons. It is noteworthy that Muriaux and collaborator have shown that RNA is an important component for retroviral particles and that some SFV RNA could be packaged into retroviral particles. Furthermore, Lebedeva et al. and Rolls et al. have shown the arising of autonomous replication replicon from SFV vectors expressing various viral glyco-proteins envelopes. Thus, these data suggested that retroviral vectors might be vehicles for all sorts of SFV derived replicons.

Experimental and modelling study of different process modes for retroviral production in a fixed bed reactor

Pseudotype vectors are promising for gene transfer in many gene therapy approaches, however, low-vector concentration in batch cultures and high temperature-dependent decay do limit sufficiently large-scale production. To overcome these obstacles, the kinetic relations of cell growth and vector formation in different culture modes need to be understood. Effective optimisation of process modes is needed to achieve sufficient yields. Experimental and modelling studies were carried out in order to analyse the impact of different process modes such as perfusion, perfused fed-batch or repeated-batch on vector titer and productivity. Retroviral pseudotype vector, derived from the murine leukaemia virus carrying the HIV-1 envelop protein MLV (HIV-1) were produced using a 200 ml fixed bed reactor for high cell density cultivation on macroporous carriers. After starting the cultivation in batch mode, the reactor was either run in perfusion, perfused fed-batch or repeated-batch. A mathematical model of the bioreaction was developed on the basis of experimental data measured in culture dishes. The ability of the model to describe all different process modes of fixed-bed cultivation without additional fitting of the parameters was proven by three long-term cultivations for more than 400 h. The results of optimisation with the aid of the model, leads to the conclusion that perfusion with optimised harvest cycles and fed flows, result in a higher yield in comparison to batch or fed batch culture.

Malignant Transformation of a PG13 Cell Line Producing an MFG Retroviral Vector Containing Wild-Type Murine Akt.

Over-expression of the pro-survival Akt gene in rat mesenchymal stem cells (MSCs) by retroviral transduction has been shown to improve cardiac repair following MSC transplant into infarcted rat heart (Mangi et al., Nature Medicine, 2003), and may hold great potential for clinical myocardial repair following cardiac damage. In preliminary transplant studies in NOD-SCID mice, we transduced human MSCs with an MFG-based retroviral vector containing genes for wild-type murine Akt and green fluorescent protein (AIG). The PG13-AIG cells used to supply retrovirus were prepared by multiple rounds of transduction of NIH 3T3-derived PG13 retrovirus packaging cells with amphotropic AIG retrovirus produced by transient transfection of the AIG vector plasmid into ϕNX-A producer cells. Primary human MSCs were transduced with PG13-AIG supernatant, were expanded in vitro for 1-3 passages, and surgically implanted after adherence to ceramic cubes into NOD-SCID mice. Three weeks after implantation of 5x106 human MSCs, a tumor mass formed around 1 of 8 ceramic cube implants, characterized histologically as a sarcoma. Retroviral insertion site analysis by linear amplification-mediated (LAM)-PCR revealed multiple insertions present within the tumor DNA. Sequence analysis of several of these vector insertion sites revealed no homology to the human genome. However, two of the insertions gave highly significant sequence homology to mouse chromosomes 15 and 11, inside introns of the mouse Dxd17 putative RNA helicase gene and mouse hypothetical Gm252 gene, respectively. These results indicate that the sarcoma was formed in greater part by Akt-transduced mouse cells rather than by transduced human MSCs. A third insertion site exhibited highly significant sequence homology to the gibbon ape leukemia virus (GALV) envelope gene present within the mouse PG13 producer cells. As GALV-pseudotyped retroviruses produced by PG13 cells do not transduce mouse cells, this argues against the tumor resulting from Akt-transduced NOD-SCID mouse cells due to production of a replication-competent retrovirus by transduced MSCs. PCR analysis detected the presence of the herpes simplex virus thymidine kinase gene (a selectable marker present in PG13 producer cells) in the tumor DNA, which together with the insertion site analysis indicates that the sarcoma was formed at least in part by Akt-transduced PG13 fibroblast cells, which may have been a rare contaminant in the MSC cell implants due to carry-over of producer cells during PG13-Akt viral supernatant transduction of MSCs. No published studies have found a tumorigenic potential for the NIH 3T3-derived PG13 cell line, and transfection of wild-type Akt into NIH 3T3 cells has been shown in several studies to confer little or no transformation or tumor-formation capacity. Regardless of origin, cell transformation by retroviral transduction of wild-type Akt, possibly with insertional mutagenesis, is a potential risk for clinical use of Akt gene transfer for enhanced stem cell survival and repair.

Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer

We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30-60% scFv(G250)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10-30% higher levels of scFv(G250)-mediated TNFalpha production and cytolysis of G250L+ RCC cells than T cells transduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Development of Zinc Finger Domains for Recognition of the 5′-CNN-3′ Family DNA Sequences and Their Use in the Construction of Artificial Transcription Factors

Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3'-, 5'-GNN-3'-, and 5'-ANN-3'-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.

Dicistronic MLV-retroviral vectors transduce neural precursors in vivoand co-express two genes in their differentiated neuronal progeny

Dicistronic MLV-based retroviral vectors, in which two IRESes independently initiate the translation of two proteins from a single RNA, have been shown to direct co-expression of proteins in several cell culture systems. Here we report that these dicistronic retroviral vectors can drive co-expression of two gene products in brain cells in vivo. Injection of retroviral vector producer cells leads to the transduction of proliferating precursors in the external granular layer of the cerebellum and throughout the ventricular regions. Differentiated neurons co-expressing both transgenes were observed in the cerebellum and in lower numbers in distant brain regions such as the cortex. Thus, we describe an eukaryotic dicistronic vector system that is capable of transducing mouse neural precursors in vivo and maintaining the expression of genes after cell differentiation.

IMPROVING ERECTILE FUNCTION BY SILENCING PHOSPHODIESTERASE-5

We investigated whether phosphodiesterase-5 (PDE5) can be down-regulated by a specific small interfering RNA (siRNA) and whether this would improve erectile function. A PDE5 siRNA encoding oligonucleotide was inserted into pSUPER-retro vector, resulting in the siRNA expressing construct, pPDE5-silencer. The construct was packaged into oncoretroviral particles and then transduced into rat cavernous smooth muscle cells (CSMCs). Cells were examined for PDE5 expression by reverse transcriptase-polymerase chain reaction, Western blotting and immunofluorescence microscopy. Cells were then treated with 10 microM sodium nitroprusside (SNP) and examined for cyclic guanosine monophosphate (cGMP) at 0, 10, 30, 60 and 240 minutes. The siRNA expressing cassette was transferred from the oncoretrovirus to lentivirus, which was then injected into rat penises. Three months later erectile function was examined by electrostimulation and PDE5 expression in cavernous smooth muscle was determined by immunohistochemistry. CSMCs transfected with pPDE5-silencer (CSMC plus siRNA) showed an 88.2% decrease in PDE5 compared with CSMCs transfected with control vector (CSMCs plus vector). Within 10 minutes of SNP treatment cells (CSMCs, CSMCs plus vector and CSMCs plus siRNA) showed similar sharp increases in cGMP. However, while cGMP levels in CSMCs and CSMCs plus vector returned to almost baseline in 1 hour, the cGMP level in CSMCs plus siRNA remained high even 4 hours after SNP treatment. Rats injected with the siRNA expressing lentivirus showed increased electrostimulated erectile function, as measured by peak intracorporeal pressure and the intracorporeal pressure increase, compared with rats injected with control lentivirus. PDE5 expression was decreased in the siRNA treated cavernous smooth muscle. PDE5 expression could be decreased by siRNA, resulting in prolonged cGMP accumulation and improved erection.

Cyclin D1 Represses p300 Transactivation through a Cyclin-dependent Kinase-independent Mechanism

Cyclin D1 encodes a regulatory subunit, which with its cyclin-dependent kinase (Cdk)-binding partner forms a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. In addition to its Cdk binding-dependent functions, cyclin D1 regulates cellular differentiation in part by modifying several transcription factors and nuclear receptors. The molecular mechanism through which cyclin D1 regulates the function of transcription factors involved in cellular differentiation remains to be clarified. The histone acetyltransferase protein p300 is a co-integrator required for regulation of multiple transcription factors. Here we show that cyclin D1 physically interacts with p300 and represses p300 transactivation. We demonstrated further that the interaction of the two proteins occurs at the peroxisome proliferator-activated receptor gamma-responsive element of the lipoprotein lipase promoter in the context of the local chromatin structure. We have mapped the domains in p300 and cyclin D1 involved in this interaction. The bromo domain and cysteine- and histidine-rich domains of p300 were required for repression by cyclin D1. Cyclin D1 repression of p300 was independent of the Cdk- and retinoblastoma protein-binding domains of cyclin D1. Cyclin D1 inhibits histone acetyltransferase activity of p300 in vitro. Microarray analysis identified a signature of genes repressed by cyclin D1 and induced by p300 that promotes cellular differentiation and induces cell cycle arrest. Together, our results suggest that cyclin D1 plays an important role in cellular proliferation and differentiation through regulation of p300.

Mice transduced with double-mutant dihydrofolate reductase-cytidine deaminase fusion gene attained protection from high dose chemotherapy

To explore the feasibility of transferring fusion gene of dihydrofolate reductase (DHFR) gene and cytidine deaminase (CD) gene into mouse bone marrow cells in order to observe the drug resistance of high dose methotrexate (MTX) and cytosine arabinoside (Ara-C) in the bone marrow cells and to improve the tolerance of myelosuppression following combination chemotherapy. Human double-mutant dihydrofolate reductase-cytidine deaminase fusion gene was transferred into two mice bone marrow cells by retroviral vector. Resistant colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in mouse bone marrow cells by retroviral infection and after treatment by drugs (Ara-C, MTX, and Ara-C + MTX). DNA was extracted from mouse bone marrow cells. The expression of drug resistant genes in mouse bone marrow cells after transferring by retroviral vector was checked by polymerase chain reaction (PCR). Bone marrow cells after coculture with the retroviral producer cells transduced with the genes (SFG-F/S-CD) showed the drug resistance colonies yield (Colony formation after exposure to Ara-C, MTX and Ara-C + MTX were 56%, 22% and 14%, respectively) and the increase in drug resistant to both MTX and Ara-C (P < 0.005). Expression of DHFR and CD gene in extracted DNA of transfected mice were demonstrated by PCR. Double drug resistant gene can not only integrate and co-express in mice bone marrow cells but also increase the drug resistance to MTX and Ara-C.