Truncated Nerve Growth Factor Receptor Research Articles

Highly Parallel Discovery of Synthetic Knockin Sequences for Enhanced Cancer Immunotherapies

Introduction T cells engineered to express transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs) have emerged as powerful treatment options for some malignancies. However, while CAR T cells have induced impressive initial response rates in patients with B-precursor acute lymphoblastic leukemia (ALL) and lymphoma, they fail to mediate long-term relapse-free survival in 40-60% of patients and have not been successful in most solid tumors. For both TCR- and CAR-based approaches, T cell persistence and long-term functionality can be diminished by chronic antigen stimulation or tonic signaling. One approach to enhance the efficacy of cell therapies that has been explored by several groups is the overexpression of specific transcription factors (e.g. AP-1/ATF transcription factors such as c-JUN or BATF); however, more systematic gain-of-function screens in human CAR T cells have not yet been done. Methods and Results Here, we introduce CRISPR-based modular pooled knockin (ModPoKI) screens to evaluate hundreds to thousands of different constructs in human T cells and their potential to overcome TCR or CAR T cell dysfunction. We generated two barcoded ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs) to identify those that confer a fitness advantage to TCR or CAR T cells. The libraries were combined with either an NY-ESO-1-specific TCR, an anti-CD19 CAR or a high affinity anti-GD2 CAR that is known to induce tonic signaling-based dysfunction. The libraries and the TCR/CAR were engineered as polycistronic sequences that were non-virally integrated into the TRAC (T cell receptor alpha chain constant) locus of primary human T cells. We performed >20 unique ModPoKI screens including bead-based single stimulation, repetitive stimulation with target cells and tonic stimulation (GD2 CAR model). While constructs containing known AP-1/ATF transcription factors BATF and BATF3 showed increased abundance across multiple screens, transcription factor AP4 (TFAP4) knockin (KI) constructs were more clearly enriched after repetitive stimulation and tonic signaling, suggesting potentially undescribed benefits in exhaustion-prone environments. We next validated single knockins of TFAP4 or a control construct (truncated nerve growth factor receptor, tNGFR) in combination with the GD2 or the CD19 CAR. TFAP4 KI led to increased levels of cytokine release, proliferation and target cell killing compared to conventional CAR T cells. Moreover, TFAP4 KI mediated increased tumor control and survival in an in vivo model. Interestingly, we observed elevated levels of CD25 (IL2RA) expression on TFAP4 KI T cells and enrichment of genes related to IL2/STAT5 pathways which could hint at a potential benefit in situations of IL-2 competition/presence of regulatory T cells. Lastly, we aimed to discover specific combinations of TFs that work in concert to enhance T cell fitness in the setting of tonic CAR signaling. We created a ~10,000-member library (102 x 102 - pairwise combinations of 100 transcription factors plus two controls) cloned into constructs with the GD2 CAR and performed a tonic signaling ModPoKI screen. Analysis of the constructs that increased the most in relative abundance highlighted that several of the top performing constructs included either TFAP4, BATF, BATF3 or a combination of TFAP4 and BATF(3), suggesting that TFAP4 and BATF(3) are key transcription factors that can coordinately drive increased T cell fitness upon repetitive stimulation. Validation analyses confirmed that the combinatorial KI of a BATF-TFAP4 construct confers highest cytotoxic capacity in vitro and in vivo compared to single knockin (BATF-RFP or RFP-TFAP4) or control knockin (RFP-tNGFR) GD2 CAR constructs. Conclusions In conclusion, ModPoKI screens allow for customizable parallel evaluation and functional characterization of hundreds to thousands of different T cell constructs. Using clinically relevant screening modalities, we nominated candidate genes that can synthetically re-write the T cell response to repetitive antigen exposure or tonic signaling and thus have the potential to program more effective TCR or CAR T cell therapies for cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Engineering Human Invariant Natural Killer T (iNKT) Cells to Overexpress Immunomodulatory Cytokines

Many studies have shown the important role that invariant natural killer T (iNKT) cells play in suppression of graft-versus-host disease (GVHD). Murine studies have also demonstrated multiple subsets of iNKT cells, including those with pro-inflammatory and immunosuppressive properties. The prevention of murine acute GVHD by the adoptive transfer of immunomodulatory iNKT cells suggests that this approach would be ideal for clinical translation. However, the heterogeneity of human iNKT cells remains poorly understood, and the uncertain ability to identify immunosuppressive human iNKT cells and expand them to clinically relevant numbers remains a limitation in clinical translation. Therefore, engineering bulk human iNKT cells to enhance immunosuppressive capacity represents a novel approach to ensuring sufficient cell numbers of immunoregulatory cells for adoptive transfer. Herein, we describe the lentiviral transduction of human iNKT cells to overexpress immunomodulatory cytokines. We focused on IL-4, because murine iNKT cell-mediated GVHD suppression is IL-4 dependent, and IL-10, because an immunosuppressive regulatory T cell-like subset of murine iNKT cells highly produces IL-10. Human iNKT cells were transduced with a lentiviral construct expressing IL-4 or IL-10 (or green fluorescent protein (GFP) as a control) and an inert truncated nerve growth factor receptor (deltaNGFR) to facilitate sorting of transduced cells. Enzyme linked immunosorbent assay revealed significantly increased expression of IL-4 or IL-10 by cells transduced with the corresponding lentivirus, as compared to untransduced, mock transduced, or GFP transduced cells, replicated with 3 iNKT donors (p<0.03) (Figure 1). This proof-of-concept data demonstrates the ability to effectively transduce human iNKT cells to overexpress immunomodulatory cytokines. Future studies will focus on evaluating the immunosuppressive capacity of these engineered cells both in vitro and in vivo (using the xenograft GVHD model). Successful suppression of GVHD by engineered iNKT cells will provide a critical step forward in identifying novel approaches to bring these promising cells to the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SELECTION OF GENOME EDITED HEMATOPOIETIC STEM CELLS PRIOR TO TRANSPLANTATION DECREASES THE ENGRAFTMENT POTENTIAL AND THERAPEUTIC EFFICACY

<h3>Background</h3> Autologous hematopoietic stem cell transplantation using genome edited cells has the potential to become a definitive therapy for lysosomal diseases, including some with neurological involvement. We have previously shown that transplantation with human, genome edited hematopoietic stem and progenitor cells (HSPC) can correct phenotypic and biochemical abnormalities in mice with mucopolysaccharidosis type I (MPSI), a multi-systemic lysosomal disorder caused by alfa-L-iduronidase (IDUA) deficiency and consequent glycosaminoglycan (GAG) storage. Before, we used GFP expression to select edited cells prior to transplantation. To effectively translate this proof-of-concept study to the clinic, we tested a clinically relevant selection marker, the truncated Nerve Growth Factor Receptor (tNGFR), which is not expressed in HSPCs, is not toxic and allows antibody-mediated cell isolation. <h3>Methods</h3> Human HSPCs were CRISPR-edited in the safe harbor CCR5 to express IDUA or IDUA and tNGFR (IDUA-tNGFR). Cells edited with IDUA were transplanted as bulk preparations while IDUA-tNGFR cells were selected with magnetic beads and transplanted as positive (IDUA-tNGFR+) and negative (IDUA-tNGFR-) fractions into NSG-MPSI mice. Phenotypical, molecular and biochemical analyses were performed 18 weeks post-transplant. <h3>Results</h3> Genome editing of IDUA-tNGFR cells was significantly lower than IDUA (14% vs. 31%), possibly due to increased IDUA-tNGFR vector size comparing to IDUA only, reducing the editing efficiency. In transplanted mice, IDUA-tNGFR+ cells engrafted poorly in hematopoietic tissues comparing to IDUA or IDUA-tNGFR- cells. The reduced engraftability and consequent lower therapeutic dose observed with tNGFR-selected cells predicted a worse performance at correcting biochemical features in the MPSI mouse model, with only few mice with detectable levels of the enzyme. Contrarily, mice transplanted with bulk cell preparations had supraphysiological levels of enzyme, with significant biochemical correction even in the brain. <h3>Conclusion</h3> These data suggest that selection of edited HSPCs for transplantation reduces drastically the therapeutic potential of the product, regardless of the selection marker. Therefore, higher therapeutic efficacy is achieved when cells are transplanted as bulk preparations (edited + unedited cells), with significant expression of the therapeutic product also in the brain, constituting a promising therapy for neurodegenerative disorders.

Effectivity Of Complement-Dependent Cytotoxicity Induced By Therapeutic Antibodies In Acute Lymphoblastic Leukemia Is Dictated By Antigen Expression Levels and Curtailed By Membrane-Bound Complement Regulatory Proteins

Membrane-bound complement regulatory proteins (mCRPs) are known to protect cells from bystander lysis and spontaneous activation of complement. In the case of antibody therapy these mCRP's may down-modulate antibody-induced complement-dependent cytotoxicity (CDC). Two of these mCRPs, CD55 (DAF) and CD59 (MIRL), have been suggested as modulators of CDC. However, no strong correlation between their expression level and target cell lysis has been demonstrated thus far. Our goal was to elucidate the role of these mCRPs in the efficacy of antibody-induced CDC.We tested the effectivity of CDC induced by the therapeutic antibodies Alemtuzumab (Alt, anti-CD52), Rituximab (Rtx, anti-CD20), and Ofatumumab (Ofa, anti-CD20) in 16 cell lines showing differential expression of target antigens and mCRPs. These cell lines were generated in our laboratory from primary acute lymphoblastic leukemia (ALL) cells and their phenotype and karyotype greatly resembles the primary malignancy. CDC was induced by incubation of target cells with a serial dilution of the respective antibody in the absence or presence of human serum for a period of 18 hours. Subsequent target cell death was determined using flow cytometry by measuring the absolute number of surviving cells (propidium iodide negative, CD19 positive) per sample, using a fixed number of CytoCount™ beads as an internal reference, and calculating the magnitude of cell loss compared to untreated control samples.A strong correlation was found between antigen expression level and CDC induction by Alt, Rtx, and Ofa. Interestingly, considerable deviation from this correlation was observed especially in cell lines with intermediate antigen expression levels. To test whether this was due to differential expression of mCRPs, surface expression levels of CD55 and CD59 were measured by flowcytometry. The magnitude of CDC strongly correlated to the mCRP expression in the cell lines with intermediate antigen expression (MFI 1128±104 (CD52, n=5) 796±132 (CD20, n=3)), resulting in 51% (Alt) and 75% (Ofa) reduction of antibody-induced lysis in cell lines with high CD55/CD59 expression compared to cell lines with low CD55/CD59 expression. In the cell lines with high antigen expression (MFI 2802±67 (CD52) 2136±692 (CD20)), the protective effect of co-expression of mCRPs appeared to be overpowered by the high target antigen expression levels.To study the individual contribution of CD55 and CD59 to the protective effect against CDC, we induced the expression of CD55 and CD59 separately in the cohort of target cell lines with intermediate target antigen expression. Cell lines were transduced, using the bicistronic LZRS vector and the ϕ-NX-A retrovirus packaging cell line, with a construct coding for CD55 or CD59, respectively, coupled to the truncated nerve growth factor receptor (tNGFR) via an internal ribosome entry site (IRES)-sequence. Cells positive for membrane expression of the tNGFR marker and CD55 or CD59 were sorted using flowcytometric cell sorting. This resulted in cell lines with significantly increased expression levels of the respective mCRP compared to their MOCK transduced counterparts, yet never exceeding physiological levels as found in the original ALL cell lines. When compared to MOCK transduced cell lines, all CD55 transduced cell lines (n=4) showed a decreased sensitivity for Alt-induced CDC with a maximum decrease of 69%. Similarly, forced induction of CD59 expression significantly decreased the sensitivity to CDC compared to MOCK transduced cell lines when using Alt (maximum decrease 52%), Rtx (maximum decrease 56%), or Ofa (maximum decrease 30%) (n=5).In conclusion, we show that efficacy of CDC induction by therapeutic antibodies is dictated by the level of target antigen expression, but is dominantly influenced by expression of the mCRPs CD55 and CD59. This restraining effect on sensitivity to antibody-induced CDC was observed using Alemtuzumab as well as Rituximab and Ofatumumab, suggesting a general mechanism independent of target antigen or type of antibody. Therefore, mCRP expression on malignant cells is likely to be an important factor in the sensitivity to antibody treatment, especially when cells with intermediate antigen expression are targeted. Disclosures:No relevant conflicts of interest to declare.

A Highly Compact Epitope-Based Marker-Suicide Gene for More Convenient and Safer T-Cell Adoptive Immunotherapy.

AbstractAbstract 1473 Background:A compact marker/suicide gene which relies on off-the-shelf reagents/pharmaceuticals would be of considerable practical utility for adoptive T-cell therapy. Marker genes enables measurement of transduction efficiency and allows sorting of transduced cells; while suicide genes allows deletion of administered T-cells in the face of toxicity. Previous marker genes include the Neomycin resistance gene, truncated nerve growth factor receptor and CD34; however these are limited by immunogenicity, unexpected biological activity and long coding region. Similarly, several suicide genes have already been described with Herpes Simplex Virus Thymidine Kinase and inducible Caspase 9 (iCasp9) in clinical use. These are limited by immunogenicity with HSVTK, limited availability of the inducing drug with iCasp9, as well as relatively long coding regions. Full-length CD20 has been tested as a suicide gene, with Rituximab as activating agent; however CD20 is biologically active when expressed in T-cells leading to spontaneous apoptosis. Aim:We sought to generate a compact marker/suicide gene construct which enables both clinical grade sorting and effective in vivo killing using off-the-shelf clinical grade reagents/pharmaceuticals – namely CD34 cliniMACS and Rituximab. By using minimal epitopes required for CD34 cliniMACS and Rituximab binding we hoped to greatly reduce the size of the construct as well as rendering it otherwise biologically inert. Results:We first sought to find the minimal epitope of CD34 which binds QBEnd10, the monoclonal antibody used in Miltenyi CliniMACS CD34 selection system. Screening a library of CD34 fragments located this to the extreme amino terminus of CD34. We further minimized this to 16 residues by sequential deletion of the amino and carboxy termini. Next, we attached this epitope to the top of the CD8alpha stalk. This highly glycosylated structure acts as an effective spacer, separating the epitope from the cell membrane but is only 49 amino acids long. We found that this construct could achieve equal binding of QBEnd10 as for full-length CD34. Next, we included a minimal epitope for rituximab binding: We first tried to co-express different fragments of the CD20 major extracellular loop identified by crystallography as the rituximab binding site. These constructs failed to bind. Next, we tried linear and circular Rituximab-binding mimetopes (described by Perosa et al. J. Immunol. 2007). Inclusion of the circular mimetope (11 amino acids) afforded excellent rituximab binding. However, complement mediated killing was poor. Further variant constructs were generated: A final construct comprising of two rituximab binding epitopes flanking a single QBEnd10 epitope on the CD8 stalk [figure (a)] was allowed optimal QBend10 and rituximab binding, as well as highly effective complement mediated killing. The binding of QBEND10 to this final construct is similar to that of full-length CD34 [figure (b)]. T-cells transduced with this construct could be effectively sorted using CD34 cliniMACS. Binding of Rituximab was 3.4 fold increased relative to native CD20 [figure (b)]. Complement mediated killing could delete >97% of transduced T-cells [figure (c)] Conclusions:We have created a 136 amino acid marker/suicide gene for T-cells. The translated protein is stably expressed on the cell surface after retroviral transduction. It binds QBEND10 with equal affinity to full length CD34. Further, the construct binds Rituximab, and the dual epitope design engenders highly effect complement mediated killing. Due to the small size of our construct, it can easily be co-expressed with typical T-cell engineering transgenes such as T-cell receptors or Chimeric Antigen Receptors and others allowing facile detection, cell selection as well as deletion of cells in the face of unacceptable toxicity with off the shelf clinical-grade reagents/pharmaceuticals. We are currently testing this construct in a murine model of GvHD. [Display omitted] Disclosures:Linch:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin-deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub-optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, together with sub-optimally loaded class I molecules. Calreticulin that lacks its C-terminal KDEL retrieval sequence assembles with the peptide-loading complex but neither retrieves sub-optimally loaded class I molecules from the cis-Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

Lentiviral Engineered T Regulatory Cells Effectively Inhibit Lymphocytes Alloreactivity across Major HLA Barriers

Adoptive immunotherapy strategies enrolling T regulatory cells (Tregs) might have a great potential in modulating donor T cells alloreactivity after Hematopoetic Cell Transplant (HCT). In murine models of HCT Tregs were shown to promote engraftment and contribute controlling graft versus host disease (GVHD) while still not conclusive data are available on humans. Ex-vivo engineering conventional CD4+ T cells to over-express the transcription factor FOXP3 is an intriguing approach to overcome the main difficulty of obtaining large amount of Tregs for experimental studies. Reports of retrovirus-mediated expression of FOXP3 not consistently resulted in functional Tregs while, recently, a lentivirus-mediated strategy was successfully reported to result in homogeneous and stable expression of FOXP3. Lentiviral transduced Tregs were able to suppress a polyclonal proliferation of CD4 purified lymphocytes stimulated with soluble Ab anti-CD3. In our study we generated lentiviral engineered Tregs (eng-Tregs) and investigated their inhibitory effect on unselected lymphocytes alloreactivity across major HLA barriers. Within the bulk lymphocytes population we separately tracked the suppressive influence of eng-Tregs on both CD4+ and CD8+ T cells. To obtain eng-Tregs, CD4+ T cells were purified from healthy donors and transduced with a bidirectional lentiviral vector encoding for FOXP3 and the truncated Nerve Growth Factor Receptor (ΔNGFR). Prior to transduction CD4+ cells were activated for 72 hours with IL2 (100U/ml), IL7 (20ng/ml) and soluble Ab anti-CD3 (200 ng/ml, only IL2 was added to the culture medium after transduction.The lentiviral transduction efficiency ranged from 8 to 25%, ΔNGFR+ T cells were positively selected and tested for their ability to suppress a mixed lymphocyte reaction across major HLA barriers. Effector peripheral blood mononuclear cells (PBMC) were collected from the same donors from whom eng-Tregs were generated. Effector PBMC were stained with CFSE in oder to separately track the alloreactive proliferation of both CD8+ and CD4+ subsets of T cells. Eng-Tregs were added on day 0 and HLA-mismatched irradiated PBMC were used as stimulators; both eng-Tregs and irradiated stimulators were used in a 1:1 ratio with the effectors. No cytokines or additional soluble stimulators were added to the MLR culture medium. The alloreactive proliferation of T cell subsets was determined by evaluating the logarithmic decrease of CFSE fluorescence intensity. The flow cytometry analysis on day +7 showed that alloreactive proliferation of both CD4+ and CD8+ effector cells was significantly inhibited (>75%) by the addition of eng-Tregs compared to controls. In order to rule out a possible role played by the naturally present Tregs (nat-Tregs), the effectors were depleted of the CD4+CD25high subpopulation before the MLR started. The observed alloreactive proliferation was higher after the depletion of nat-Tregs but still it could be significantly inhibited by the addition of eng-Tregs. Eng-Tregs did not significantly expanded when cultured in vitro (up to 2 weeks) with IL2 (100U/ml) but maintained a stable expression of the transgene and retained their suppressive capacity. Our data show that lentiviral engineered Tregs can efficiently down-modulate both CD4+ and CD8+ T cell alloreactivity across major HLA barriers. The observed independence from the presence of nat-Tregs might be important in future experimental HCT settings where the adoptive infusion of eng-Tregs might encounter a great variability in the number and activity of recipient's nat-Tregs. The possibility of transducing a potentially unlimited number of CD4+ cells makes this strategy appealing for future pre-clinical studies to control GVHD in HCT settings.

Clinical-scale selection of anti-CD3/CD28-activated T cells after transduction with a retroviral vector expressing herpes simplex virus thymidine kinase and truncated nerve growth factor receptor.

Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.

Retroviral transfer and expression of human MDR-1 in a murine haemopoietic stem cell line does not alter factor dependence, growth or differentiation characteristics.

In view of the recent report of a myeloproliferative syndrome in mice that had received an MDR-1-transduced haemopoietic graft, we have investigated the potential effects of MDR-1 expression on primitive haemopoietic cell growth and differentiation. Retroviral gene transfer was used to achieve exogenous expression of either MDR-1 or truncated nerve growth factor receptor (tNGFR) in the multipotent murine haemopoietic progenitor cell line, FDCP-mix. Following gene transfer, clonal lines were derived and FACS analysis confirmed appropriate expression of each transgene. MDR-1 (but not tNGFR) expression was associated with verapamil-sensitive rhodamine efflux and resistance to killing by etoposide. When growth factor responsiveness, proliferative capacity and differentiation capacity were examined, MDR-1 expressing FDCP-mix cells exhibited a normal phenotype and mimicked the response of tNGFR-expressing or untransduced FDCP-mix cells. Thus, in the model system we have used, MDR-1 does not perturb haemopoietic cell growth and development and our data do not support a myeloproliferative role for MDR-1.

Increased transduction efficiency of primary hematopoietic cells by physical colocalization of retrovirus and target cells.

Efficient gene transfer into hematopoietic stem cells offers a number of potential therapeutic applications. However, the relatively low titer of retroviral supernatants and the requirement for cell division to ensure integration have meant that transduction efficiency has been low. We have modified a flowthrough approach to cell transduction and have been able consistently to increase gene transfer efficiency into human hematopoietic progenitor cells. We transduced CD34 cells with retroviral vectors encoding a truncated nerve growth factor receptor (NGFR) or neo. Retroviral supernatant was pulled through 0.2-micron polycarbonated membranes, followed by placement of cells on the filter. In the absence of cytokines, the transduction efficiency of CD34 cells with a NGFR vector was increased 3-11-fold over that obtained at an identical MOI in liquid culture to produce 11%-44% transduction. Furthermore, both Thy1+ and Thy1- subsets in a total CD34 population were transduced with similar efficiency, and transduction with a neo vector, as measured by G418 resistance in clonogenic assays, increased 1.5-5-fold. The mechanism by which gene transfer is improved may reflect colocalization of cells and retrovirus. Costaining of cells transduced on the filter with an NGFR retrovirus with both an NGFR antibody and a gp70 antibody that recognizes viral coat protein revealed high-level coexpression. The levels of in vitro gene transfer we obtain are equivalent to those observed when CD34 cells are cocultured in liquid culture with cytokines. However, culture with cytokines may commit CD34 cells to differentiation and has produced disappointingly low levels of subsequent in vivo gene transfer. Gene marking studies using distinguishable retroviral vectors will provide a means of learning whether the effects of flowthrough transduction genuinely enhance the efficiency of gene transfer to human marrow-repopulating cells.

Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells. We compared levels of expression in PA317 fibroblasts, F9 (EC) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors based on our previously reported MND backbone, which has alterations to address three viral elements implicated as repressors of expression by Mo-MuLV: the enhancer, the primer binding site, and the negative-control region. Expression was evaluated with three reporter genes, the chloramphenicol acetyltransferase (CAT) gene, whose expression was measured by enzymatic assay and by Northern blotting; a truncated nerve growth factor receptor (tNGFR), whose expression was measured by fluorescence-activated cell sorting (FACS) as a cell surface protein; and the enhanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher levels of CAT activity (5- to 300-fold) and greater quantities of vector-specific transcripts in ES and EC cells transduced with the modified MND-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT-SN are >80 times more abundant than the L-CAT-SN transcripts. FACS analysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-tNGFR-SN, indicated that only 1.04% of the CCE cells containing the L-tNGFR-SN vector expressed the cell surface reporter, while the MND-tNGFR-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cells containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99.89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Essentially identical results were produced with an analogous pair of vectors encoding EGFP. In unselected pools of F9 cells 48 h posttransduction, the L-EGFP-SN vector drove expression in only 5% of the population while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion of cells showing expression from L-EGFP-SN decreased slightly to 3% while expression from the MND-EGFP-SN vector persisted in 80% of the cells. Interestingly, in the few ES and EC cells which did show expression from the L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN vector in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector, MND, achieves higher net levels of expression due to a greater frequency of expression, which may be useful for the expression of exogenous genes in EC and ES cells.

Generation of the truncated form of the nerve growth factor receptor by rat Schwann cells. Evidence for post-translational processing.

These studies were initiated to determine whether the soluble, truncated form of the nerve growth factor (NGF) receptor arises from post-translational processing of the intact, membrane-bound receptor or from an alternatively spliced mRNA. Pulse-chase analysis of cultured primary rat Schwann cells coupled with immunoprecipitations using antibodies to the intracellular and extracellular domains of the receptor were used to monitor receptor production. Three forms of the NGF receptor (80, 83, and 85 kDa) displaying a precursor product relationship were detected over the 2-h chase period; only the 85-kDa species was detected on the cell surface. Truncated receptors (50 and 52 kDa) were detected in conditioned media 5 h after cell labeling but were never observed intracellularly. Polymerase chain reaction and RNase protection analyses of NGF receptor mRNA targeted toward the coding region for the transmembrane domain detected no splice variants that could generate truncated receptor, and media conditioned by fibroblasts transfected with rat receptor cDNA, in which splicing cannot occur, nonetheless contained the truncated receptor protein. Taken together, these results suggest that the truncated NGF receptor does not arise as a distinct translation product but rather from a post-translational modification of the intact, surface-bound form of the protein.

Evidence for endocytosis-dependent proteolysis in the generation of soluble truncated nerve growth factor receptors by A875 human melanoma cells

We have identified nerve growth factor receptor (NGFR) on the cell surface and a truncated nerve growth factor receptor (NGFRt) in the conditioned medium of NGFR-negative cells that have been transfected with either the gene or the cDNA for the full-length receptor. By using cell surface iodination or metabolic labeling of A875 human melanoma cells, coupled with immunoprecipitation, we have determined the half-life of the cell-associated receptor to be approximately 7 h. Concomitant with receptor degradation is the accumulation of NGFRt in the extracellular medium. Approximately one-fifth of the labeled receptor can be recovered as the truncated species. These data support the hypothesis that NGFRt is generated by proteolysis of previously intact receptor. Furthermore, although no specific protease inhibitor assayed could affect this processing, NGFR degradation and truncation were retarded by treatment with: 1) the weak base amines, ammonium chloride or methylamine; 2) the carboxylic ionophore, monensin; or 3) the vacuolar ATPase inhibitor, bafilomycin A1, all agents that dissipate endosomal/lysosomal proton gradients via alternate mechanisms. Incubation of cells at 4 degrees C precluded NGFR degradation and truncation. The presence of ligand did not alter the time course of receptor truncation.

Developmental regulation of human truncated nerve growth factor receptor

Monoclonal antibodies (designated XIF1 and IIIG5) recognizing distinct epitopes of the human truncated nerve growth factor receptor (NGF-Rt) were used in a two-site radiometric immunosorbent assay to monitor levels of NGF-Rt in human urine as a function of age. Urine samples were collected from 70 neurologically normal subjects ranging in age from 1 month to 68 years. By using this sensitive two-site radiometric immunosorbent assay, NGF-Rt levels were found to be highest in urine from 1-month old subjects. By 2.5 months, NGF-Rt values were half of those seen at 1 month and decreased more gradually between 0.5 and 15 years. Between 15 and 68 years, urine NGF-Rt levels were relatively constant at 5% of 1-month values. No evidence for diurnal variation of adult NGF-Rt was apparent. Pregnant women in their third trimester showed significantly elevated urine NGF-Rt values compared with age-matched normals. Affinity labeling of NGF-Rt with 125I-NGF followed by immunoprecipitation with ME20.4-IgG and gel autoradiography indicated that neonatal urine contained high amounts of truncated receptor (Mr = 50 kd); decreasingly lower amounts of NGF-Rt were observed on gel autoradiograms with development, indicating that the two-site radiometric immunosorbent assay correlated well with the affinity labeling technique for measuring NGF-Rt. NGF-Rt in urines from 1-month-old and 36-year-old subjects showed no differences in affinities for NGF or for the monoclonal antibody IIIG5. These data show that NGF-Rt is developmentally regulated in human urine, and are discussed in relation to the development and maturation of the peripheral nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain

The expression of nerve growth factor (NGF) receptor in adult rat brain was studied by immunohistochemistry with a specific anti-rat NGF receptor monoclonal antibody, 192-IgG. Intense NGF receptor immunoreactivity (NGFRI) was found in structures known to be NGF responsive, including forebrain cholinergic neurons in medial septum, diagonal band of Broca, and basal nucleus of Meynert; central processes of neural-crest-derived sensory ganglion neurons and their innervated nucleus also contained such immunoreactivity. Distinct NGFRI staining was also found in many brain areas and cell types not known to be NGF responsive, including some hypothalamic regions, circumventricular organs, some areas related to the optic system, olfactory glomeruli, ependymal and subependymal cells in some locations, mesencephalic nucleus of the trigeminal nerve, cerebellar molecular layer, central linear nucleus, solitary tract and its nucleus, and inferior olive. The NGFRI in the circumventricular organs was further studied by in vivo labeling of 125I-ligands. Intravenously injected 125I-NGF, but not 125I-cytochrome c, was specifically accumulated in the area postrema. Biochemical study of the NGF receptor showed a major band of molecular weight of approximately 90 KDa in the area postrema, choroid plexus, median eminence, and medial septum with the relative content consistent with that seen by immunohistochemistry. No evidence of a truncated NGF receptor was observed. The results of this study suggest that NGF and its receptor have broader roles in adult mammalian brain than previously thought.